NZ712059A - Compositions and methods for controlling head blight disease - Google Patents

Abstract

Disclosed is an isolated microbial strain of Bacillus sp. strain SGI-015-F03 (deposited as NRRL B-50760) and pesticidally active variants thereof, wherein said microbial strain has suppressive activity against head blight disease. Also disclosed is a method for preventing, inhibiting or treating the development of Fusarium graminearum, wherein said method comprises growing the above-described microbial strain in a growth medium or soil of a host plant prior to or concurrent with host plant growth in said growth medium or soil.

Description

COMPOSITIONS AND METHODS FOR CONTROLLING HEAD BLIGHT DISEASE INCORPORATION OF SEQUENCE LISTING The materialin the accompanying Sequence g is hereby incorporated by reference in its entirety. The accompanying file, named “SG11520—1WO_ST25.txt”, was created on July 24, 2012 and is 55 Kb. The file can be accessed using Microsoft Word on a er that uses Window OS.

FIELD OF THE INVENTION The present invention relates to the biological control of phytopathogenic diseases.

Specifically, it relates to compositions and methods useful for controlling head blight disease in cereal , such as Wheat and barley.

BACKGROUND OF THE ION Head blight, also known as head scab, pink mold, Whiteheads and tombstone scab, is a devastating disease ing wheat, barley, and several other cereal crops ide, ularly in USA, Europe and China. This disease can reach epidemic levels and cause extensive damage to grains, especially wheat and barley in humid and semi-humid cereal growing areas of the world, ing India, Russia, France, Germany, and the United Kingdom. Particularly, wheat scab or head blight is one of the more damaging es of wheat in the United States. Nationwide, this e has caused the Wheat industry millions of dollars in yield losses. In the Midwest and High Plains, wheat scab is the major obstacle to wheat production in recent years. Head blight in addition to attacking Wheat also attacks and reproduces on barley, oats, corn, and many other cereals.

This serious plant disease can be caused by several phytopathogens, but primarily by several s in the flmgal genus Fusarz'um. For example, causal agents of the head blight disease in wheat include a number of different Fusarium species, e. g. F. culmorum, F. graminearum (teleomorph, Gibberella zeae), F. eum (teleomorph, G. avenacea), F. poae, as well as by non-Fusarium pathogens such as Microdochium nivale (teleomorph, Monographella nivalz's), and Microdochium majus. In the United States, Europe and most agronomically important areas of the world, the predominant causative agent of head blight is Fusarium graminearum (teleomorph, Gibberella zeae sensu strict).

These pathogens typically survive on plant debris. They invade and damage the spikelets of the grain head during flowering, thus preventing or lly impeding the development of grain in the grain head. As a result, the invading scab pathogen can either kill part of the grain head or the entire grain head. Some infected seeds are low in vigor and often fail to germinate. Infected seeds that germinate often die early in the ng stage due to crown rot or root rot, g poor stands in the following crop plant. Healthy seedlings can also become infected at emergence. In addition to poor, unthrifty stand, yield losses due to pathogen infestation can be quite high if conditions are favorable for development of the disease.

The fungal pathogens ofFusarz‘um genus spread across grain cultivating areas all over the world, and are known to cause severe damage, particularly in areas With high rainfall between flowering and grain filling. When Fusarium graminearum is the causative agent, this e is of primary concern because it does not only reduce the cial values of the contaminated grains, in addition to yield losses, but because Fusarium infection can also lead to the accumulation oftrichothecene mycotoxins in the grains thus threatening the health ofhuman and livestock. Trichothecenes are major mycotoxin contaminants of cereals worldwide, causing feed refusal, vomiting, ea and weight loss in non-ruminant s and posing a health threat to other s and humans when exposure levels are high. This threat has been exacerbated by the recent shift in the F. graminearum strains in the United States towards greater toxin production and vigor. Most ntly found mycotoxins are deoxynivalenol (DON, also known as vomitoxin) and zearalenone (ZEA). Deoxynivalenol in ular is a very dangerous toxin, causing gastrointestinal disorders accompanied by hemorrhagic conditions and the like in humans and animals that eat infected grains, leading to death in some cases. Since deoxynivalenol is generally stable against changes in pH and high ature, detoxification can be very difficult. Therefore, grains contaminated beyond a certain level cannot be used in any form ofbrewing, processing, or livestock feed, and thus often need to be disposed of.

To date, various strategies have been deployed for controlling head blight in crop plants. Promising options include chemical measures, the development of resistant crop ars, and traditional practices of crop rotation and tillage of fields. Among these s, chemical pesticides can be somewhat effective in reducing head blight infestation, but residue concerns regarding the use of fungicides late in crop development, typically at flowering stages only a few weeks before harvest, lessen their attractiveness. Advances in developing resistant cultivars using traditional breeding and genetic engineering represent another disease control alternative are also occurring. Reported examples of c engineering advances include altering the production of a plant hormone or manipulating the plant hormone ing pathway. In recent years, considerable advances in the area of traditional breeding have been made in understanding the genetic basis of resistance to head blight disease and a number of genes and quantitative trait loci (QTL) conferring resistance have been reported. However, progress in improving crop resistance to head blight disease has been slow, largely because ofthe difficulty of studying this disease. In fact, relatively little is currently tood about the mechanisms involved in resistance or susceptibility. In addition, the c diversity ofFusarz'um species, which are the predominant ive agents of the disease, often raises concerns regarding how durable the efficacy of chemical ides and resistant cultivars will be. As a result, practically all wheat cultivars currently in large-scale production remain vulnerable to infection.

In addition, although some success in controlling head blight disease can be expected by traditional practices such as plowing fields to bury crop residues infested with causative agent, 6. g. F. gramz'nearum, after t, conventional tillage of fields after harvesting is not compatible with the soil conservation practice mum tillage.

Considering the ial of long distance inoculum dispersal and the diverse crops that can act as alternative hosts of the pathogens, crop rotation is often an ble solution. In addition to the problem of pesticide residues in the environment, reports of pesticide ance and instances ofDON t increases in grain can also be concerns with their use. Further, costs and increasing concerns in the public and private sectors over pesticide residues in the environment and food product safety render this disease control alternative less attractive, and have led to requests for crop cultivation using as little ides as possible.

In summary, despite considerable advances in developing techniques to control head blight, reducing the impact of this devastating disease on grain production and quality remains an intractable problem. ore, identification and development ofnew head blight controlling ques is essential in improving the tion and quality ofmany cereal crops. These problems require urgent solution not only in United States, but also across the globe, including the Asia and Europe. ical control ofhead blight disease has attracted considerable interest since the mid . Biological control agents (BCAs), though currently very limited in , could be an environmentally acceptable method for ntially decreasing the level of disease incited by ens such as um. Public acceptance, compatibility with other disease management measures, and durability are among favorable factors in support of developing strategies for ically controlling head blight disease. ical control agents could play an important role in organic cereal production. In conventional tion, such agents may extend protection of spikes past the flowering stage after chemical fungicides can no longer be applied. To date, significant advances in the area of biological control have been ed. For example, certain strains of spore-producing bacteria (such as Bacillus and Pseudomonas species) and yeasts (such as Cryptococcus species) show some promise for the control of head blight disease and the ion of mycotoxin contamination.

However, despite these and other advances, the need s for improved microorganisms for use in the biological control of head blight disease. Although BCAs have become a more acceptable solution for plant pathogens and BCA products have been marketed to a greater extent than ever before, to date there have been few attempts to develop gies and nistic microorganism for biologically controlling head blight disease. Furthermore, the life cycle ofFusarz'um spp. and other causative agents of head blight disease suggest that the pathogens can potentially be susceptible to biocontrol techniques using antagonistic microorganisms at different developmental stages. Thus, there is a need to identify new biological control agents, preferably with ent modes of actions, as well as biocontrol methods that can help effectively prevent or suppress the development ofhead blight disease.

SUMMARY OF THE INVENTION Compositions comprising microbiological strains and cultures are provided herein.

Certain strains, cultures, and itions thereof are useful for the control of head blight disease, for example, of various crop plants including wheat and other cereal plants.

Biological control compositions, and methods ofuse thereof to prevent, inhibit or treat the development of plant pathogens or disease and for preserving plant yield, are also provided.

Also provided are methods for the use of such compositions as biological control agents in ation with other agriculturally effective compounds or compositions for controlling l plant pathogens.

In one aspect, the present invention provides isolated microbial strains having suppressive activity against head blight disease. The microbial strains in accordance to this aspect of the present invention are selected from the group consisting of the genera Microbacterium, Bacillus, Mycosphaerella, and Variovorax. In some preferred embodiments, the microbial strains are selected from the group consisting of Mycosphaerella sp. strain SGI H11 (deposited as NRRL 50471), Microbacterium sp. strain SGI C06 (deposited as NRRL B-50470), Microbacterium sp. strain SGIG08, Variovorax sp. strain SGIG01 (deposited as NRRL B-50469), us sp. strain SGIF03 (deposited as NRRL B-50760), Bacillus sp. strain SGIH06 (deposited as NRRL B- 50761), and pesticidally active variants of any thereof. T he microbial strain in accordance to some other preferred embodiments can comprise a DNA sequence that exhibits at least 85% sequence identity to any one of the nucleotide sequences in the Sequence Listing. r provided are biologically pure es and enriched cultures of the microbial strains disclosed .

Also provided in another aspect of the present invention are itions that se a microbial strain of the invention or a culture thereof, and an agriculturally effective amount of a compound or composition selected from the group consisting of an acaricide, a bactericide, a ide, an insecticide, a microbicide, a nematicide, a pesticide, and a fertilizer. The compositions in some embodiments of this aspect may be prepared as a formulation selected from the group consisting of an emulsion, a colloid, a dust, a granule, a pellet, a , a spray, an emulsion, and a solution. In some other embodiments, the compositions may be provided with a carrier. In some preferred embodiment, the r is an agriculturally acceptable carrier. In some particularly preferred embodiment, the carrier is a plant seed. In other preferred ments, the composition is a seed coating ation.

Further ed in the present disclosure are seeds that are coated with a composition according to the present invention.

In another aspect of the present invention, there are provided s for preventing, inhibiting or treating the development of a plant pathogen. T he methods involve growing a microbial strain of the invention or a culture thereof in a growth medium or soil of 2012/048012 a host plant prior to or concurrent with host plant growth in the growth medium or soil. In some preferred embodiments of this aspect, the plant pathogen causes head blight disease. In some particularly preferred ments, the plant en is Fusarium graminearum.

In yet another aspect ofthe present invention, methods are provided for preventing, inhibiting or treating the development of head blight e of a plant. The methods e applying to the plant, or to the plant's surroundings, an effective amount of a microbial strain ofthe invention or a culture thereof. In one embodiment, such head blight disease is caused by the fungus Fusarz'um gramz’nearum. In some embodiments ofthis aspect, the microbial strain or a culture thereof is d to soil, a seed, a root, a flower, a leaf, a portion of the plant, or the whole plant. In a preferred embodiment, the plant is susceptible to Fusarz'um graminearum. In some other preferred embodiments, the plant is wheat plant, a corn plant, a barley plant, or an oat plant. In another preferred embodiment, the ial strain of the present invention or a culture thereof is established as an endophyte on the plant.

Another further aspect of the invention provides non-naturally occurring plants.

The non-naturally occurring plants are artificially infected with a ial strain of the invention or a culture thereof. Further provided in some red embodiments of this aspect are seed, uctive tissue, vegetative tissue, plant parts, and progeny of the non- naturally occurring plants.

Yet another aspect of the invention provides a method for preparing an agricultural composition. The method involves inoculating the microbial strain according to the present invention or a culture thereof into or onto a substratum and allowing it to grow at a temperature of 1-37°C until obtaining a number of cells or spores of at least 102-103 per milliliter or per gram.

These and other objects and features of the invention will become more fully apparent from the following detailed description of the invention and the claims.

DETAILED DESCRIPTION OF THE ION Some definitions Unless otherwise defined, all terms of art, notations and other scientific terms or ology used herein are ed to have the meanings commonly understood by those of skill in the art to which this invention pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art. Many of the techniques and procedures described or referenced herein are well understood and commonly employed using conventional methodology by those skilled in the art.

The singular form “a”, “an”, and “the” include plural references unless the context y dictates otherwise. For example, the term “a cell” includes one or more cells, including mixtures thereof. By "comprising" is meant including, but not limited to, er follows the word "comprising".

The expressions onistic microorganism” and “microbial antagonist” are used herein interchangeably in reference to a microorganism is intended to mean that the subject strain exhibits a degree of inhibition of head blight disease exceeding, at a statistically significant level, that of an ted control.

Antibiotic: the term “antibiotic”, as used herein, refers to any substance that is able to kill or inhibit the growth of a microorganism. Antibiotics may be produced by any one or more of the following: 1) a microorganism, 2) a synthetic process, or 3) a semisynthetic process. An antibiotic may be a microorganism that secretes a volatile organic compound.

Furthermore, an antibiotic may be a volatile organic compound secreted by a microorganism. icidal: the term “bactericidal”, as used herein, refers to the ability of a composition or substance to increase mortality or inhibit the growth rate of bacteria.

Inhibition of the growth rate of bacteria can be commonly quantified as the reduction of viable bacterial cells over time.

Biological control: the term “biological control” and its abbreviated form “biocontrol”, as used , is d as control of a pathogen or insect or any other rable sm by the use of at least a second organism other than man. An example of a known mechanism ofbiological control is the use of microorganisms that control root rot by out-competing fungi for space on the surface of the root, or rganisms that either inhibit the growth of or kill the pathogen. The “host plant” in the t of biological control is the plant that is tible to disease caused by the pathogen. In the context of ion of an organism, such as a fungal species, from its natural environment, the “host plant” is a plant that supports the growth ofthe fungus, for example, a plant of a species the fungus is an endophyte of.

The term "cereal" as used herein is intended to refer to any cereal species that can be susceptible to head blight disease. Cereals reported to be susceptible include wheat, barley, oats, and triticale, though wheat and barley are the two crops in which this disease presents a significant economic problem.

An "effective amoun " refers to an amount ent to affect beneficial or desired results. In terms of e management, treatment, inhibition or protection, an effective amount is that amount sufficient to suppress, stabilize, reverse, slow or delay progression of the target infection or e states. As such, the expression “an effective amoun ” is used herein in reference to that quantity of antagonist treatment which is necessary to obtain a reduction in the level of pathogen development and/or in the level of pathogenic disease relative to that ing in an untreated control. Typically, an effective amount of a given antagonist treatment provides a reduction of at least 20%; or more typically, between 30 to 40%; more typically, n 50—60%; even more typically, between 70 to 80%; and even more typically, between 90 to 95%, relative to the level of e and/or the level of pathogen development occurring in an untreated control under suitable conditions of treatment. An effective amount can be administered in one or more administrations. The actual rate of application of a liquid ation will usually vary from a minimum of about 1 X 103 to about 1 X 1010 Viable cells/mL and preferably from about 1 X 106 to about 5 X 109 Viable cells/mL. Under most conditions, the antagonistic microbial s ofthe ion described in the Examples below, would be optimally effective at application rates in the range of about 1 X 106 to l X 109 Viable cells/mL, assuming a mode of application which would achieve substantially uniform contact of at least about 50% of the plant tissues. If the antagonists are applied as a solid formulation, the rate of application should be controlled to result in a able number of viable cells per unit area ofplant tissue surface as obtained by the aforementioned rates of liquid ent. Typically, the biological control agents of the present ion are biologically effective when delivered at a concentration in excess of 106 CFU/g (colony forming units per gram), preferably in excess of 107 CFU/g, more preferably 108 CFU/g, and most preferably at 109 CFU/g.

Further, the sion "effective microbial antagonist" used herein in nce to a microorganism is intended to mean that the subject microbial strain exhibits a degree of tion of head blight disease exceeding, at a statistically cant level, that of an untreated control. Typically, an effective microbial antagonist has the ability to effect a reduction of at least 20%; or more typically, between 30 to 40%; more typically, between 50- 60%; even more typically, between 70 to 80%; and even more typically, between 90 to 95%, relative to the level of disease and/or the level ofpathogen development occurring in an untreated control under suitable conditions of treatment.

Composition: A “composition” is intended to mean a combination of active agent and at least another compound, carrier or composition, inert (for example, a detectable agent or label or liquid carrier) or active, such as a pesticide.

Isolated microbial strain, isolated culture, biologically pure culture, and enriched culture: As used herein, the term “isolated” as applied to a microorganism (e.g., bacterium or microfungus) refers to a microorganism which has been removed and/or purified from an environment in which it naturally occurs. As such, an “isolated strain “of a microbe as used herein is a strain that has been removed and/or purified from its natural milieu. Thus, an “isolated microorganism” does not include one residing in an environment in which it naturally occurs. Further, the term ted" does not necessarily reflect the extent to which the e has been d. A "substantially pure culture" of the strain of microbe refers to a culture which contains ntially no other microbes than the desired strain or strains of microbe. In other words, a ntially pure culture of a strain of microbe is substantially free of other contaminants, which can e microbial contaminants as well as undesirable chemical contaminants. Further, as used herein, a "biologically pure" strain is intended to mean the strain separated from als with which it is normally associated in nature. Note that a strain associated with other strains, or with compounds or materials that it is not normally found with in , is still defined as "biologically pure." A monoculture of a particular strain is, of course, "biologically pure." As used herein, the term "enriched culture" 2012/048012 of an ed ial strain refers to a microbial culture that contains more than 50%, 60%, 70%, 80%, 90%, or 95% of the isolated strain.

As used herein, an "endophyte" is an endosymbiont that lives within a plant for at least part of its life Without causing apparent disease. Endophytes may be transmitted either vertically (directly from parent to offspring) or horizontally (from individual to unrelated individual). Vertically-transmitted fungal endophytes are typically asexual and transmit from the maternal plant to offspring via fungal hyphae penetrating the host's seeds. Bacterial endophytes can also be transferred vertically from seeds to seedlings (Ferreira et al., 2008).

Conversely, horizontally-transmitted endophytes are typically sexual, and transmit via spores that can be spread by Wind and/or insect vectors. Endophytes of crop plants have been receiving considerable attention with t to their ability to control both disease and insect infestation, as well as promoting plant growth. onally comparable protein: The phrase “functionally comparable n” as used herein describes those proteins that have at least one characteristic in common. Such characteristics include sequence similarity, biochemical activity, transcriptional pattern similarity and ypic activity. Typically, the functionally comparable proteins share some ce similarity or at least one biochemical. Within this definition, homologs, orthologs, paralogs and analogs are considered to be functionally comparable. In addition, functionally comparable proteins generally share at least one biochemical and/or phenotypic activity. Functionally comparable ns will give rise to the same characteristic to a similar, but not necessarily the same, degree. Typically, functionally comparable proteins give the same characteristics where the quantitative measurement due to one ofthe comparables is at least 20% of the other; more typically, between 30 to 40%; more lly, between ; even more typically, between 70 to 80%; even more typically, between 90 to 95%; even more typically, between 98 to 100% ofthe other.

Fungicidal: As used herein, “fungicidal” refers to the ability of a composition or substance to decrease the rate of growth of fimgi or to increase the ity of fimgi.

Fusarz'um fungus: For purposes of this ion it is understood that the use of term Fusarium fimgus is intended to include both the sexual (teleomorphic) stage of this organism and also the asexual rphic) stage, also referred to as the perfect and imperfect fungal , respectively. For example, the anamorphic stage ofFusarium graminearum is ella zeae, a causative agent ofhead blight disease. This disease typically occurs when the flower or seed head becomes inoculated with conidia produced by the imperfect form or ascospores produced by the perfect form of this fimgus.

Mutant: As used herein, the term “mutant” or “varian ” in reference to a microorganism refers to a modification ofthe parental strain in which the desired biological activity is similar to that expressed by the parental strain. For example, in the case of Mcrobacterium the “parental strain” is defined herein as the original Microbacz‘erium strain before mutagenesis. s or ts may occur in nature without the intervention of man. They also are obtainable by treatment with or by a variety of methods and compositions known to those of skill in the art. For example, a parental strain may be treated with a chemical such as N-methyl-N'-nitro-N—nitrosoguanidine, ethylmethanesulfone, or by irradiation using gamma, X-ray, or UV-irradiation, or by other means well known to those practiced in the art.

Nematicidal: The term “nematicidal”, as used , refers to the ability of a substance or composition to increase mortality or inhibit the growth rate of nematodes.

Pathogen: The term gen" as used herein refers to an organism such as an alga, an arachnid, a bacterium, a fungus, an insect, a nematode, a parasitic plant, yeast, a protozoan, or a virus capable of producing a disease in a plant or animal. The term "phytopathogen" as used herein refers to a pathogenic organism that infects a plant.

Percentage of percent ty: “percentage of sequence ty”, as used herein, is determined by comparing two lly locally aligned ces over a comparison Window defined by the length of the local alignment between the two sequences. The amino acid sequence in the comparison window may comprise additions or deletions (e. g., gaps or overhangs) as compared to the reference sequence (which does not comprise ons or deletions) for l alignment of the two sequences. Local alignment between two ces only includes segments of each sequence that are deemed to be sufficiently similar according to a criterion that depends on the algorithm used to perform the alignment (6. g.

BLAST). The percentage identity is calculated by determining the number of ons at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number ofmatched ons by the total number of positions in the window of comparison and lying the result by 100. Optimal alignment of sequences for comparison may be ted by the local homology algorithm of Smith and Waterman (1981) Add. APL. Math. 2:482, by the global homology alignment algorithm ofNeedleman and Wunsch (J. Mol. Biol. 48:443, 1970), by the search for similarity method of Pearson and Lipman (Proc. Natl. Acad. Sci. USA 85: 2444, 1988), by tic implementations of these algorithms (NCBI BLAST, WU-BLAST, BLAT, SIM, BLASTZ), or by tion. Given that two sequences have been identified for comparison, GAP and BESTFIT are preferably employed to determine their optimal alignment. lly, the default values of 5.00 for gap weight and 0.30 for gap weight length are used.

The term “substantial sequence identity” between polynucleotide or polypeptide sequences refers to polynucleotide or polypeptide comprising a sequence that has at least 50% sequence identity, preferably at least 70%, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, even more preferably at least 95%, and most preferably at least 96%, 97%, 98% or 99% sequence identity compared to a reference sequence using the programs.

Query c acid and amino acid sequences can be searched t subject nucleic acid or amino acid sequences residing in public or proprietary databases. Such searches can be done using the National Center for Biotechnology Information Basic Local Alignment Search Tool (NCBI BLAST v 2.18) program. The NCBI BLAST program is ble on the intemet from the National Center for Biotechnology Information .ncbi.nlm.nih.gov/Blast.cgi). Typically the following ters for NCBI BLAST can be used: Filter options set to "default”, the Comparison Matrix set to "BLOSUM62”, the Gap Costs set to “Existence: 11, Extension: 1”, the Word Size set to 3, the Expect (E threshold) set to 16-3, and the minimum length of the local alignment set to 50% of the query sequence length. Sequence identity and similarity may also be determined using GenomeQuestTM software (Gene-IT, Worcester Mass. USA).

The term “pesticidal”, as used herein, refers to the ability of a substance or composition to decrease the rate of growth of a pest, i.e., an red organism, or to increase the mortality of a pest.

By “suppressive activity” of a biological control agent against a phytopathogen is meant the ability of the agent to suppress, inhibit, stabilize, reverse, slow, or delay the development ofthe pathogen itself, or the progression of the infection or disease states caused by the pathogen.

Variant: A “varian ”, as used herein in reference to a microorganism, is a strain having identifying characteristics of the species to which it belongs, While having at least one nucleotide sequence variation or fiably different trait with respect to the parental strain, where the trait is genetically based (heritable). For e, for a Microbacterium sp. SGI- SGI—Ol4-CO6 strain having fungicidal activity, identifiable traits include 1) the ability to suppress the growth ofFusarium graminearum and its teleomorph Gibberella zeae; 2) the ability to suppress the development of head blight disease; 3) having housekeeping genes with greater than 95%, greater than 96%, greater than 97%, greater than 98%, or greater than 99% sequence identity to the housekeeping genes ofMicrobacterz'um sp. SGI-Ol4-CO6 can be used to confirm a variant as Microbacterium sp. 4-C06.

For nucleic acids and polypeptides, the term “varian ” is used herein to denote a ptide, protein or polynucleotide molecule with some differences, generated synthetically or naturally, in their amino acid or nucleic acid sequences as compared to a reference ptide or polynucleotide, respectively. For example, these differences include substitutions, insertions, deletions or any desired ations of such changes in a reference or ptide. , polypeptide Polypeptide and protein variants can further consist of changes in charge and/or post-translational ations (such as glycosylation, methylation, phosphorylation, etc.) All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

No ion is made that any reference constitutes prior art. The discussion of the references states What their authors assert, and the applicants reserve the right to challenge the cy and ence of the cited documents. It will be clearly tood that, although a number of prior art publications are referred to herein, this reference does not tute an admission that any of these documents forms part of the common general knowledge in the art. 2012/048012 Methods for Taxonomic Identification Microorganisms can often be distinguished based on direct microscopic analysis (do all of the cells in a sample look the same on examination), staining characteristics, simple molecular is (such as a simply restriction fiagment length polymorphism (RFLP) ination), and so forth. In addition to the illustrative examples of such taxonomic analysis techniques as described at Examples 2-3 of the present disclosure, taxonomic identification of a microorganism can e up to several different levels of analysis, and each analysis can be based on a different characteristic of the organism. Such taxonomic analyses can include nucleic acid-based analysis (e.g., analysis of individual specific genes, either as to their presence or their exact sequence, or expression of a ular gene or a family of genes), protein-based analysis (e. g. , at a fimctional level using direct or indirect enzyme assays, or at a structural level using irnmuno—detection techniques), and so forth. a. Nucleic acid-based analysis: One of ordinary skill in the art will appreciate that a wide variety of nucleic acid-based techniques are known and can be useful in ing the taxonomic identification of a given microorganism. These techniques can be used to identify cells by gene sequence or to identify cells that have particular genes or gene families.

Common gene families useful for taxonomic studies e 168 gene family, actin gene family, and inase A (recA) gene family. These methods typically e amplifying and sequencing genes from very small numbers of cells, and therefore often overcome the problems of concentrating cells and their DNA from dilute suspensions. The term “nucleic acid amplification" generally refers to techniques that increase the number of copies of a nucleic acid molecule in a sample or specimen. Techniques useful for nucleic acid amplification are well known in the art. An example of nucleic acid amplification is the rase chain reaction (PCR), in which a biological sample collected from a subject is contacted with a pair of ucleotide primers, under conditions that allow for the hybridization of the primers to nucleic acid template in the sample. The primers are extended under suitable ions, dissociated fi'om the te, and then re-annealed, extended, and dissociated to y the number of copies of the nucleic acid. Other examples of in vitro amplification techniques include strand displacement amplification; transcription-free isothermal amplification; repair chain reaction amplification; ligase chain reaction; gap filling ligase chain reaction amplification; coupled ligase detection and PCR; and RNA transcription-free amplification.

In addition to the illustrative example primers provided herein, see, e.g. Examples 2-3 and the Sequence Listing, primers have also been designed ely, and new ones are ually being designed, for individual species or phylogenetic groups of microorganisms.

Such narrowly targeted primers can be used with the methods described herein to screen and/or identify specifically only the microorganisms of interest.

Methods for preparing and using nucleic acid primers are described, for example, in Sambrook et al. (In lar Cloning: A Laboratory Manual, CSHL, New York, 1989), Ausubel et al. (ed.) (In Current Protocols in Molecular y, John Wiley & Sons, New York, 1998). Amplification primer pairs can be derived from a known sequence, for example, by using computer programs intended for that purpose such as Primer (Whitehead Institute for ical Research, Cambridge, Mass). One of ry skill in the art will appreciate that the specificity of a particular probe or primer increases with its length. Thus, for example, a primer comprising 30 consecutive nucleotides of an rRNA-encoding tide or flanking region thereof will anneal to a target sequence With a higher specificity than a corresponding primer of only 15 nucleotides. Thus, in order to obtain greater specificity, probes and s can be selected that comprise at least 20, 25, 30, 35, 40, 45, 50 or more consecutive tides of a target tide sequence such as the 168 rRNA.

Common techniques for the preparation of nucleic acids useful for nucleic acid applications (6. g. , PCR) include phenol/chloroform tion or use of one of the many DNA extraction kits that are available on the market. Another way that DNA can be amplified is by adding cells directly to the c acid amplification reaction mix and relying on the denaturation step of the amplification to lyse the cells and release the DNA.

The product ofnucleic acid amplification reactions may be further characterized by one or more of the standard techniques that are well known in the art, including electrophoresis, restriction endonuclease cleavage patterns, oligonucleotide ization or ligation, and/or nucleic acid sequencing. When in hybridization techniques are used for cell identification purposes, a variety ofprobe labeling methods can be useful, including cent labeling, radioactive labeling and non-radioactive labeling. When nucleic acid sequencing techniques are used, homology search for the nucleotide sequence of the amplified nucleic acid molecules can be conducted using various ses ofknown sequences, including but not d to DDBJ/GenBank/EMBL databases. b. Protein—based analysis: In addition to analysis of nucleic acids, microorganisms can be mically characterized and identified based on the presence (or absence) of specific proteins directly. Such analysis can be based on the activity of the specified protein, e. g. , through an enzyme assay or by the response of a co-cultured organisms, or by the mere presence ofthe specified n (which can for instance be determined using immunologic methods, such as in situ immunofluorescent antibody staining).

Enzyme assays: By way of example, fluorescent or chromogenic ate analogs can be included into the growth media (e.g. microtiter plate cultures), followed by incubation and screening for reaction products, thereby identifying cultures on a basis of their enzymatic activities.

Co-cultivation se: In some embodiments of the present invention, the activity of an enzyme carried by a microbial isolate can be assayed based on the response (or degree of response) of a co-cultured organism (such as a reporter organism).

A variety ofmethods can also be used for identifying microorganisms selected and isolated from a source nment by binding at least one antibody or antibody—derived le to a molecule, or more ularly an epitope of a molecule, ofthe microorganism.

Antijrrricroorganism protein antibodies may be produced using standard procedures described in a number of texts, including Harlow and Lane (Antibodies, A Laboratory Manual, CSHL, New York, 1988). The determination that a particular agent binds substantially only to a protein of the desired microorganism may readily be made by using or adapting routine ures. One suitable in vitro assay makes use of the Western blotting procedure (described in many standard texts, including Harlow and Lane (Antibodies, A tory Manual, CSHL, New York, 1988)). r fragments of antibodies (antibody-derived molecules, for instance, FAbs, Fvs, and single-chain Fvs (SCFvs)) can also serve as c binding agents. Methods of making these fragments are routine. ion of antibodies that bind to cells on an array ofthis invention can be carried out using standard techniques, for instance ELISA assays that e a detectable signal, for instance a fluorescent or scent signal.

Isolated cultures of the invention As described in more details in the Examples section of the present disclosure, Applicants have discovered several agriculturally beneficial novel microorganisms, for example, effective suppressors ofhead blight disease. ularly, these novel antagonistic microorganisms are ive for reduction of head blight severity and for inhibition of the growth ofFusarium graminearum, a primary ive agent of head blight disease in wheat.

The microbial antagonists were identified from a pool of approximately 5,000 microbial strains obtained from Wild plant samples collected from l locations in the United States.

Initial selection of antagonistic microorganism was based on the ability of the microorganisms to suppress the development of F. graminearum pathogen and that of its teleomorph Gibberella zeae in an in vitro antagonism assay. Selected microbial antagonists were then bio-assayed in a greenhouse study on wheat ngs, which involved inoculation of the ngs with the microbial strains, followed by repeated ations of F. graminearum , for the ability of the microbial s to reduce the severity of fungal infestation and for their ability to ve seed yield. The antagonistic microorganisms selected in this manner were found to be effective in reducing the severity of head blight in greenhouse trials.

Taxonomic analysis further determined that each ofthe antagonistic microorganisms described in the present disclosure are closely related either the bacterial genus Microbacterium, the bacterial genus Bacillus, the bacterial genus Variovorax, or the fungus genus Mycosphaerella.

The genus Microbacterium, the type genus of the family Microbacteriaceae, is generally ered to accommodate Gram—positive, non-spore forming, rod-shaped bacteria that were originally ed during early studies on lactic acid-producing bacteria. Members ofthe genus Microbacterium are originally characterized largely by their marked heat resistance, presence in dairies, and production of small amounts of I..(+) lactic acid from glucose. In contrast to other genera of the family Microbacterz'aceae in which species are characterized by a coherent peptidoglycan type, species ofMicrobacterium possess omithine or lysine either in the inter-peptide bridge or at position 3 of B-type peptidoglycan. In other chemotaxonomic properties, such as isoprenoid quinones (MK-l l, MK-12, , polar lipids, fatty acids and base composition of DNA, members of the genus exhibit the usual range of diversity found in other genera of Microbacteriaceae. Two bacterial genera Microbacterium and Aureobacterz'um can be united according to some taxonomy studies. To date, members ofthe genus acterium comprise at least 33 s, which have been isolated from a broad range of habitats, including soil, dairy products, plant galls, insects or clinical specimens. Various aspects of their ecology, phylogeny, taxonomy, culture methods, and long-term preservation conditions have recently been summarized by Evtushenko and Takeuchi (2006). One of skill in the art will readily appreciate that microorganism of the genus acterium can be taxonomically identified y by any ofthe taxonomic identification techniques described above, including the chemotaxonomic analysis of the cell wall peptidoglycan and comparative 16S rDNA sequence analysis as described in Evtushenko and Takeuchi (2006) and references cited therein, as well as in those described in Examples 2-3 ofthe present disclosure. As discussed in detail below, to date l naturally occurring rganisms have been ed as having antagonistic activity against head blight disease. r, there are no reports prior to the present invention that be a microorganism ofthe genus Mycobacterium having such antagonistic activity.

Further, prior to the t ion, the present inventors were not aware of any s or processes of using a bacterial strain of the genus Mycobacterz'um as trol agent in preventing, inhibiting or treating the development of a causative pathogen of head blight disease.

The genus Bacillus is a genus of Gram-positive/variable, spore-forming, rod- shaped bacteria. Similarly to other genera associated with the early history of microbiology, such as Pseudomonas or Vibrz’o, nearly 266 species members of the Bacillus genus are found ubiquitously, and it is widely considered to be one of the genera with the largest 168 diversity and environmental diversity. Bacillus species can be obligate aerobes or facultative anaerobes, and test positive for the enzyme catalase. Ubiquitous in nature, Bacillus includes both free—living and pathogenic species. Under stressful environmental conditions, the cells produce oval endospores that can stay dormant for extended periods. These characteristics originally defined the genus, but not all such species are closely related, and many have been moved to other . In fact, several studies have tried to reconstruct the phylogeny of the genus. The Bacillus-specific study with the most diversity covered is by Xu and Cote [Int]. J ofSyst. Evol. Microbiol. 53 (3): 695—704; 2003], using 16S and the ITS region, where they divide the genus into 10 groups, which includes the nested genera Paenibacillus, Brevibacillus, Geobacillus, Marinibacillus and Virgibacillus. However, according to more recent studies, the genus Bacillus contains a very large number of nested taxa and especially in both 168 and 23S it is considered to be paraphyletic to Lactobacillales (Lactobacillus, Streptococcus, lococcus, Listeria, eta), due to Bacillus coahuilensis and others (see, e. g., Yarda et al., Syst. Appl. Microbiol. 31 (4): 241—250, 2008; Yarda et al., Syst. Appl.

Microbiol. 33 (6): 291—299, 2010]. One particular clade, formed by B. anthracis, B. cereus, B. mycoz'des, B. pseudomycoz'des, B. gz‘ensis and B. weihenstephanensis under current classification standards, should be a single species (within 97% 168 identity), but due to medical reasons, they are considered separate species. In addition to the taxonomy is methods described at Examples 2-3 ofthe present disclosure, the phylogenetic and taxonomic analyses ofBacillus species can be performed by a variety oftechniques, including those discussed in s in Xu and Cote, 2003; Yarda ez‘ al., 2008; Yarda et al., 2010..

The genus Variovorax was originally created by reclassification ofAlcaligenes paradoxus as Variovorax paradoxus (Willems er al., 1991), which is widely consider as the type species of this genus. V. xus has been extensively studied as a model for novel biodegradation agents, as well as microbe/microbe and microbe/plant interactions. Other s include V. ensis, V. soli (Kim et al., 2006), and V. borom’cumulans (Miwa et al., 2008). Variovorax species are catabolically very diverse and engage in mutually beneficial interactions with other ial species in many biodegradations, and therefore s ecological importance and high application potential. For example, a soil methanotroph, only when tured together with a V. paradoxus strain, exhibits high affinity for methane (a potent greenhouse gas), and this trait is not y observed in laboratory es. Similarly, a close relative of Variovorax has been found to be the central, non—photosynthetic partner within the phototrophic consortium "Chlorochromatz'um aggregatum.” Some species of the genus orax also have the ability to interfere with the communication of other bacteria. Some yet other species of the genus Variovorax can intimately interact with other biota (e.g. , plants) in various ecosystems. Moreover, some Variovorax species, residing in the area just outside plant roots and/or inside a plant, have been reported for being capable of promoting plant growth via the reduction of ethylene levels, the repression of quorum-sensing-controlled enesis, and the increase of resistance to heavy metals, which greatly benefits phytoremediation. In addition to the my analysis methods described at Examples 2-3 ofthe present disclosure, the phylogenetic and taxonomic analyses of Variovorax species can be performed by a variety of techniques, including those discussed in details elsewhere herein. haerella is a very large fiingus genus, with over 2,000 s names and at least 500 species associated with more than 40 anamorph genera (especially Cercospora, Pseudocercospora, Septoria, Ramularia, etc). In addition, several thousand phs lack telomorphs. The genus of Mycosphaerella es species that are pathogens, saprobes, endophytes, or mutualistic associations. Various aspects of their ecology, phylogeny, taxonomy, culture methods, and long-term preservation ions have recently been reported (see, e. g. Crous et al., nia, 23:119-146,2009). One of skill in the art will readily appreciate that microorganism ofthe genus Mycosphaerella can be taxonomically fied by any ofthe taxonomic techniques described above, or a ation thereof.

Most common techniques include comparative ce analyses using 16S rDNA sequences and the internal transcribed spacer regions as described in, for example, Crous et al.

, Studies in Mycology, 55:235—253,2006; Crous et al., 2009, supra; Goodwin et al., Phytopathology 91: 648—65 8, 2001; and those described in Examples 2—3 of the present disclosure.

Deposit of ical Material Purified cultures of microbial strains identified as having suppressive activity against head blight disease were deposited in the Agricultural Research e Culture Collection located at 1815 N. University Street, Peoria, IL 61604, USA (NRRL) in accordance with the Budapest Treaty for the purpose nt procedure and the regulations thereunder (Budapest Treaty). Accession numbers for these deposits are as follows: N ——— SGI—OlO-Hll NRRL 50471 SGIC06 NRRL 13—50470 SGIG01 llllilNRRL B-50469 SGI-OlS-FO3 NRRL B-50760 SGI-OlS-H06 NRRL B-50761 The microbial s have been deposited under conditions that ensure that access to the culture will be available during the pendency of this patent application to one determined by the Commissioner of Patents and Trademarks to be ed thereto under 37 C.F.R. §1.14 and 35 U.S.C. §122. The ts ent substantially pure cultures of the ted strains. The deposits are ble as ed by foreign patent laws in countries wherein counterparts of the subject application or its progeny are filed. However, it should be understood that the availability of a deposit does not constitute a license to practice the subject invention in derogation of patent rights d by governmental action.

Preferred microorganisms of the present invention have all ofthe identifying characteristics of the deposited strains and, in ular, the identifying characteristics of being able to suppress the development of head blight disease as described herein, and as being able to suppress the development ofFusarz‘um gramz'nearum pathogen and its teleomorph Gibberella zeae as described herein. In particular, the preferred microorganisms ofthe present invention refer to the deposited microorganisms as described above, and mutants thereof.

Microbiological compositions The microbiological compositions ofthe present invention that se isolated microbial strains or es thereof can be in a variety of forms, including, but not d to, still cultures, whole cultures, [stored stocks of cells, mycelium and/or hyphae (particularly glycerol stocks), agar strips, stored agar plugs in glycerol/water, freeze dried stocks, and dried stocks such as lyophilisate or mycelia dried onto filter paper or grain seeds. As defined elsewhere herein, “isolated culture” or grammatical equivalents as used in this disclosure and in the art is understood to mean that the ed to culture is a culture fluid, pellet, scraping, WO 16361 dried sample, lyophilate, or section (for e, hyphae or mycelia); or a t, container, or medium such as a plate, paper, filter, matrix, straw, pipette or pipette tip, fiber, needle, gel, swab, tube, vial, particle, etc. that contains a single type of organism. In the present invention, an isolated e of a microbial antagonist is a culture fluid or a scraping, pellet, dried ation, lyophilate, or n of the microorganism, or a support, container, or medium that contains the microorganism, in the absence of other organisms.

The present disclosure further provides compositions that contain at least one isolated microbial strains or cultures thereof of the present invention and a carrier. The carrier may be any one or more of a number of carriers that confer a variety of properties, such as increased stability, wettability, dispersability, etc. Wetting agents such as natural or synthetic surfactants, which can be nonionic or ionic surfactants, or a combination thereof can be ed in a composition ofthe invention. Water-in—oil emulsions can also be used to formulate a composition that includes at least one isolated microorganism of the present invention (see, for example, US. Patent No. 7,485,451, incorporated by reference herein).

Suitable formulations that may be prepared include wettable powders, granules, gels, agar strips or s, thickeners, and the like, ncapsulated particles, and the like, liquids such as aqueous flowables, aqueous suspensions, water-in—oil emulsions, etc. The formulation may e grain or legume products (e.g. or beans, broth or flour , ground grain derived from grain or beans), starch, sugar, or oil. The carrier may be an agricultural carrier.

In certain preferred embodiments, the carrier is a seed, and the composition may be applied or coated onto the seed or d to saturate the seed.

In some embodiments, the agricultural carrier may be soil or plant growth medium.

Other agricultural carriers that may be used include water, fertilizers, plant-based oils, ants, or combinations thereof. Alternatively, the agricultural r may be a solid, such as diatomaceous earth, loam, , alginate, clay, bentonite, vermiculite, seed cases, other plant and animal products, or combinations, including granules, pellets, or sions.

Mixtures of any of the aforementioned ingredients are also contemplated as carriers, such as but not limited to, pesta (flour and kaolin clay), agar or flour-based pellets in loam, sand, or clay, etc. Formulations may include food sources for the cultured organisms, such as barley, rice, or other biological materials such as seed, plant parts, sugar cane bagasse, hulls or stalks from grain processing, ground plant material (e.g. “yard waste”) or wood from building site refuse, sawdust or small fibers from recycling of paper, fabric, or wood. Other suitable formulations will be known to those skilled in the art.

In the liquid form, 6. g. solutions or suspensions, the microorganisms may be mixed or suspended in water or in aqueous solutions. Suitable liquid diluents or carriers include water, aqueous solutions, petroleum lates, or other liquid carriers.

Solid compositions can be prepared by dispersing the antagonist microorganisms in and on an appropriately d solid carrier, such as peat, wheat, bran, vermiculite, clay, talc, bentonite, diatomaceous earth, fuller's earth, pasteurized soil, and the like. When such formulations are used as wettable s, biologically compatible dispersing agents such as non—ionic, anionic, amphoteric, or cationic dispersing and emulsifying agents can be used.

In a preferred embodiment, the compositions contemplated herein prevent attack by head blight disease upon plants, particularly cereal plants, such as wheat, barley, oat, and corn and, when used in sufficient amounts, to act as microbial antagonists. These compositions, similarly to other biocontrol agents, have a high margin of safety because they typically do not burn or injury the plant.

As bed in great detail throughout the present disclosure, control of head blight disease may be effected by application of one or more ofthe iological compositions ofthe present invention to a host plant or parts ofthe host plant. The compositions can be applied in an amount effective to reduce the level of head blight relative to that in an untreated control. The active constituents are used in a concentration sufficient to inhibit plant pathogen pment ofthe targeted plant pathogen when applied to the cereal plant. As will be apparent to a d person in the art, effective concentrations may vary depending upon such factors as: (a) the type of the plant or ltural commodity; (b) the physiological condition ofthe plant or agricultural commodity; (c) the tration of ens affecting the plant or agricultural commodity; (d) the type of disease injury on the plant or ltural commodity; (e) weather conditions (e.g. temperature, humidity); and (f) the stage of plant disease. According to the invention, typical concentrations are those higher than 1 X 102 CFU/mL of carrier. Preferred concentrations range from about 1 X 104 to about 1 X 109 CFU/mL, such as the trations ranging from 1 X 106 to 1 X 108 CFU/mL. More red concentrations are those of from about 35 to about 150 mg dry WO 16361 microbial mass per gram of carrier (dry formulation) or per milliliter of carrier (liquid composition). In solid formulations, the rate of application should be controlled to result in a comparable number of viable cells per unit area of plant tissue surface as obtained by the aforementioned rates of liquid treatment. Typically, the ical control agents of the present invention are biologically effective when delivered at a tration in excess of 106 CFU/g (colony forming units per gram), preferably in excess of 107 CFU/g, more preferably 108 CFU/g, and most preferably at 109 CFU/g.

In some embodiments, the amount of one or more ofthe ical l agents ' in the microbial compositions ofthe present invention can vary depending on the final formulation as well as size or type of the plant or seed utilized. Preferably, the one or more biological control agents in the microbial compositions are present in about 2% W/w/ to about 80% w/w ofthe entire formulation. More preferable, the one or more biological control agents employed in the compositions is about 5% W/W to about 65% w/w and most preferably about 10% w/w to about 60% w/w by weight ofthe entire formulation.

As it will be appreciated by those skilled in the art, the microbiological compositions of the invention may be applied to the Wheat plant or other cereals using a variety of conventional methods such as dusting, coating, injecting, g, rolling, dipping, spraying, or ng, or any other riate technique which does not significantly injure the wheat plant or other cereals to be treated. Particularly preferred is the spray method.

Typically, the compositions ofthe invention are chemically inert; hence they are ible with substantially any other constituents of the spray schedule. They may also be used in combination with biologically compatible pesticidal active agents as for example, ides, nematicides, fungicides, insecticides, and the like. They can also be used in combination with plant grth affecting substances, such as fertilizers, plant growth regulators, and the like, provided that such compounds or substances are biologically compatible.

When used as pesticides or fungicide in their commercially available formulations and in the use forms, prepared from these formulations, the active microbial antagonists and compositions according to the present invention can furthermore be present in the form of a mixture with synergists. Synergists are compounds by which the activity of the active compositions is increased without it being necessary for the synergist added to be active When used as pesticides in their commercially available formulations and in the use forms, prepared from these formulations, the active microbial nists and compositions according to the invention can furthermore be present in the form of a mixture with inhibitors which reduce the degradation of the active compositions after application in the habitat of the plant, on the surface of parts of plants or in plant tissues.

The active microbial antagonists and compositions according to the invention, as such or in their formulations, can also be used as a mixture with known acaricides, bactericides, fungicides, icides, microbicides, nematicides, pesticides, or combinations thereof, for e in order to Widen the spectrum of action or to t the development of resistances in this way. In many cases, synergistic effects result, 2'. e. the activity of the mixture can exceed the activity of the individual components. A mixture with other known active compounds, such as fertilizers, growth regulators, safeners and/or semiochemicals is also contemplated.

In a preferred embodiment ofthe present invention, the compositions may further include at least one chemical or biological pesticide. The amount of at least one chemical or biological pesticide employed in the compositions can vary ing on the final formulation as well as the size of the plant and seed to be treated. Preferably, the at least one chemical or biological pesticide employed is about 0.1% w/w to about 80% w/w based on the entire formulation. More preferably, the at least one chemical or biological pesticide is present in an amount of about 1% w/w to about 60% w/w and most preferably about 10% w/w to about 50% w/w.

A variety of ides is apparent to one of skill in the art and may be used.

Exemplary chemical ides include those in the carbamate, organophosphate, organochlorine, and prethroid classes. Also included are chemical control agents such as, but not limited to, benomyl, borax, captafol, , chorothalonil, ations containing copper; formulations ning dichlone, an, iodine, zinc; fungicides that inhibit ergosterol biosynthesis such as but not limited to blastididin, cymoxanil, fenarimol, flusilazole, folpet,, imazalil, ipordione, maneb, manocozeb, metalaxyl,oxycarboxin, utanil, oxytetracycline, PCNB, pentachlorophenol, prochloraz, propiconazole, quinomethionate, sodium aresenite, sodium DNOC, sodium hypochlorite, sodium phenylphenate, streptomycin, sulfur, tebuconazole, terbutrazole, thiabendazolel, thiophanate— methyl, triadimefon, tricyclazole, triforine, validimycin, zolin, zineb, and ziram. A variety of insecticidal compounds can be useful for the compositions ofthe invention, including but not limited to those cited in U.S. Pat. Appl. No. 201 32A1.

The microbiological compositions ofthe present ion preferably include at least one biological pesticide. Exemplary biological pesticides that are suitable for use herein and can be included in a microbiological composition according to the present ion for preventing a plant pathogenic disease include microbes, animals, ia, fungi, genetic material, plant, and natural products of living organisms. In these compositions, the microorganism ofthe present invention is isolated prior to formulation with an additional organism. For example, microbes such as but not limited to species ofAmpelomyces, Aureobasidium, Bacillus, Beauverz'a, Candida, Chaetomium, Cordyceps, Cryptococcus, Dabaryomyces, Erwim'a, Exophilia, Gliocladium, Mariannaea, Paecilomyces, Paem'bacz'llus, Pantoea, Pichia, Pseudomonas, olomyces, myces, and derma can be provided in a composition with the nistic microorganisms of the present invention, with fungal strains of the Muscodor genus being particularly red. Use of the microbiological compositions according to the present invention in combination with the microbial nists disclosed in US Patent No. 7,518,040; US Patent No. 7,601,346; US Patent No. 6,312,940 is also particularly preferred.

Examples of fungi that can be combined with microbial antagonists and compositions ofthe present invention in a ition include, without limitation, or species, Aschersonia aleyrodz's, Beauverz'a bassiana ("white ine"), Beauveria brongniartii, Chladosporium herbarum, Cordyceps clavulata, Cordyceps entomorrhiza, Cordycepsfacis, Cordyceps gracilz's, Cordyceps melolanthae, Cordyceps militaris, Cordyceps myrmecophz'la, Cordyceps ravenelz'i, Cordyceps sinensis, Cordyceps cephala, Cordyceps subsessz'lis, Cordyceps unilateralz's, Cordyceps variabilis, Cordyceps washingtonensis, Culicinomyces clavosporus, Entomophaga g‘ryllz', Entomophaga maimaiga, Entomophaga muscae, Entomophagapraxibulli, phthoraplutellae, Fusarz'um laterz'tium, Hirsutella citriformz's, Hirsutella thompsom', izium anisopliae ("green ine"), Metarhizz'umflaviride, Muscodor albus, Neozygitesfloridana, Nomuraea rileyz', Paecilomycesfarinosus, Paecz'lomycesfumosoroseus, Pandora z'dz's, Tolypocladz'um cylindrosporum, Verticillz'um lecam'z', Zoophthora radicans, and mycorrhizal species such as Laccarz'a r. Other mycopesticidal species will be apparent to those skilled in the art.

The present invention also provides methods of treating a plant by application of any of a y of customary formulations in an effective amount to either the soil (1'. e. , in- furrow), a portion of the plant (1'. e. , drench) or on the seed before planting (i.e., seed coating or dressing). Customary formulations include solutions, emulsifiable concentrate, wettable powders, suspension concentrate, soluble powders, granules, sion-emulsion trate, natural and synthetic materials impregnated with active compound, and very fine control release capsules in ric substances. In certain embodiments of the present invention, the biological control compositions are formulated'in powders that are available in either a ready-to-use formulation or are mixed together at the time of use. In either embodiment, the powder may be admixed with the soil prior to or at the time of planting. In an ative ment, one or both of either the biological control agent or insect control agent is a liquid formulation that is mixed together at the time of treating. One of ordinary skill in the art understands that an effective amount of the ive compositions depends on the final formulation of the composition as well as the size of the plant or the size of the seed to be treated.

Depending on the final formulation and method of application, one or more suitable additives can also be introduced to the compositions of the t invention.

Adhesives such as carboxymethylcellulose and l and synthetic polymers in the form of powders, es or latexes, such as gum arabic, chitin, polyvinyl alcohol and polyvinyl acetate, as well as natural phospholipids, such as ins and lecithins, and synthetic phospholipids, can be added to the present compositions.

In a preferred embodiment, the iological compositions are formulated in a single, stable solution, or emulsion, or suspension. For solutions, the active chemical compounds (i. e., the pest control agents) are typically dissolved in solvents before the biological control agent is added. Suitable liquid solvents include eum based aromatics, such as xylene, toluene or alkylnaphthalenes, aliphatic hydrocarbons, such as cyclohexane or paraffins, f0r example petroleum fractions, mineral and vegetable oils, alcohols, such as butanol or glycol as well as their ethers and esters, ketones, such as methyl ethyl ketone, methyl isobutyl ketone or cyclohexanone, strongly polar solvents, such as ylformamide and dimethyl sulphoxide. For emulsion or suspension, the liquid medium is water. In one embodiment, the chemical control agent and biological control agent are suspended in separate liquids and mixed at the time of ation. In a preferred embodiment of suspension, the insect control agent and biologic are combined in a ready-to- use formulation that exhibits a shelf-life of at least two years. In use, the liquid can be sprayed or can be applied foliarly as an atomized spray or row at the time of planting the crop. The liquid composition can be introduced to the soil before germination of the seed or directly to the soil in contact with the roots by utilizing a y of techniques known in the art including, but not limited to, drip irrigation, sprinklers, soil injection or soil drenching.

Optionally, stabilizers and buffers can be added, including alkaline and alkaline earth metal salts and organic acids, such as citric acid and ascorbic acid, inorganic acids, such as hydrochloric acid or sulfuric acid. Biocides can also be added and can include formaldehydes or formaldehyde- releasing agents and derivatives of c acid, such as xybenzoic acid.

Seed coating formulations In some particularly preferred embodiments, the biocontrol compositions of the present invention are formulated as a seed treatment. The seed treatment preferably comprises at least one insect control agent and at least one biological control agent. It is plated that the seeds can be substantially mly coated with one or more layers of the trol compositions sed herein using conventional methods of mixing, spraying or a combination thereof through the use of treatment application equipment that is specifically designed and ctured to accurately, safely, and efficiently apply seed ent products to seeds. Such equipment uses various types of coating technology such as rotary s, drum coaters, fluidized bed techniques, spouted beds, rotary mists or a combination thereof. Liquid seed treatments such as those of the present invention can be applied Via either a spinning "atomizer" disk or a spray nozzle which evenly distributes the seed treatment onto the seed as it moves though the spray pattern. Preferably, the seed is then mixed or tumbled for an additional period oftime to achieve additional treatment distribution and . The seeds can be primed or unprimed before coating with the inventive compositions to increase the uniformity of germination and nce. In an alternative embodiment, a dry powder formulation can be metered onto the moving seed and allowed to mix until completely distributed.

Another aspect ofthe invention provides seeds treated with the subject microbiological compositions. One embodiment provides seeds having at least part ofthe surface area coated with a microbiological composition ing to the present invention. In a specific embodiment, the microorganism-treated seeds have a spore concentration or microbial cell concentration from about 106 to about 109 per seed. The seeds may also have more spores or microbial cells per seed, such as, for example 1010, 1011 or 1012 spores per seed. The microbial spores and/or cells can be coated freely onto the seeds or, preferably, they can be formulated in a liquid or solid composition before being coated onto the seeds.

For example, a solid composition comprising the microorganisms can be ed by mixing a solid carrier with a suspension of the spores until the solid carriers are impregnated with the spore or cell suspension. This mixture can then be dried to obtain the desired particles.

In some other embodiments, it is contemplated that the solid or liquid biocontrol compositions of the present invention further contain functional agents e of protecting seeds from the harmful effects of selective herbicides such as activated carbon, nutrients (fertilizers), and other agents capable of improving the germination and quality of the products or a combination thereof.

Seed coating methods and compositions that are known in the art can be particularly useful when they are d by the addition of one of the embodiments ofthe present invention. Such g methods and apparatus for their application are disclosed in, for e, U.S. Pat. Nos. 5,918,413; 5,554,445; 5,389,399; 945; and 4,465,017.

Various seed coating itions are disclosed, for example, in U.S. Pat. Appl. Nos.

US20110033432, US20100154299, U.S. Pat. Nos. 5,939,356; 5,876,739, 5,849,320; 084, 5,661,103; 544, 5,328,942; 4,735,015; 4,634,587; 4,372,080, 4,339,456; and 4,245,432, among others. ' A variety of additives can be added to the seed treatment formulations comprising the inventive itions. Binders can be added and include those composed preferably of 2012/048012 an ve polymer that can be natural or synthetic without phytotoxic effect on the seed to be coated. The binder may be selected from polyvinyl acetates; polyvinyl acetate copolymers; ethylene vinyl e (EVA) copolymers; polyvinyl alcohols; polyvinyl alcohol mers; celluloses, including ethylcelluloses, methylcelluloses, hydroxymethylcelluloses, hydroxypropylcelluloses and carboxymethylcellulose; polyvinylpyrolidones; polysaccharides, including , modified starch, dextrins, maltodextrins, alginate and chitosans; fats; oils; proteins, ing gelatin and zeins; gum arabics; shellacs; Vinylidene chloride and dene chloride copolymers; calcium lignosulfonates; c copolymers; nylacrylates; polyethylene oxide; acrylamide polymers and copolymers; polyhydroxyethyl acrylate, methylacrylamide monomers; and polychloroprene.

Any of a variety of colorants may be employed, including organic chromophores classified as nitroso; nitro; azo, including monoazo, bisazo and polyazo; acridine, anthraquinone, azine, diphenyhnethane, indamine, indophenol, e, e, ocyanine, thiazine, thiazole, triarylmethane, xanthene. Other additives that can be added include trace nutrients such as salts of iron, manganese, boron, copper, cobalt, molybdenum and zinc. A polymer or other dust control agent can be applied to retain the treatment on the seed surface.

In some specific embodiments, in addition to the microbial cells or spores, the coating can fiirther comprise a layer of adherent. The adherent should be non-toxic, biodegradable, and adhesive. Examples of such materials include, but are not limited to, polyvinyl acetates; polyvinyl acetate copolymers; polyvinyl alcohols; polyvinyl alcohol copolymers; celluloses, such as methyl celluloses, hydroxymethyl celluloses, and hydroxymethyl propyl oses; dextrins; alginates; sugars; molasses; polyvinyl pyrrolidones; polysaccharides; proteins; fats; oils; gum arabics; gelatins; syrups; and starches.

More examples can be found in, for e, US. Pat. No. 7,213,367 and US. Pat. Appln.

No. US20100189693.

Various additives, such as adherents, dispersants, surfactants, and nutrient and buffer ients, can also be included in the seed treatment formulation. Other conventional seed treatment additives include, but are not limited to, coating agents, wetting , buffering agents, and polysaccharides. At least one agriculturally acceptable carrier can be added to the seed treatment formulation such as water, solids or dry powders. The dry powders can be derived from a variety ofmaterials such as calcium carbonate, gypsum, vermiculite, talc, humus, activated charcoal, and various phosphorous compounds.

In some embodiment, the seed coating composition can comprise at least one filler which is an organic or inorganic, natural or synthetic component with which the active components are combined to facilitate its application onto the seed. Preferably, the filler is an inert solid such as clays, natural or synthetic silicates, silica, resins, waxes, solid fertilizers (for example ammonium salts), natural soil minerals, such as s, clays, talc, lime, quartz, attapulgite, montmorillonite, ite or diatomaceous , or synthetic minerals, such as silica, alumina or silicates, in particular um or magnesium silicates.

The seed treatment formulation may further include one or more ofthe following ingredients: other pesticides, including nds that act only below the ground; fimgicides, such as captan, thiram, metalaxyl, fludioxonil, oxadixyl, and isomers of each of those materials, and the like; herbicides, including compounds selected from sate, carbamates, rbamates, acetamides, triazines, dinitroanilines, glycerol ethers, zinones, s, phenoxys, ureas, and c acids; herbicidal safeners such as benzoxazine, benzhydryl derivatives, N,N—diallyl dichloroacetamide, various dihaloacyl, oxazolidinyl and thiazolidinyl compounds, ethanone, naphthalic anhydride compounds, and oxime derivatives; fertilizers; and biocontrol agents such as other naturally-occmring or recombinant bacteria and fungi from the genera Rhizobium, Bacillus, Pseudomonas, Serratia, Trichoderma, Glomus, Gliocladium and hizal fungi. These ingredients may be added as a separate layer on the seed or alternatively may be added as part of the seed coating composition ofthe invention.

Preferably, the amount of the novel composition or other ients used in the seed ent should not inhibit ation of the seed, or cause phytotoxic damage to the seed.

The microorganism-treated seeds may also be further enveloped with a film overcoating to protect the coating. Such overcoatings are known in the art and may be applied using fluidized bed and drum film coating techniques.

WO 16361 In principle, any plant seed capable of germinating to form a plant that is susceptible to attack by nematodes and/or pathogenic fungi can be treated in accordance with the invention. Suitable seeds include those of cereals, coffee, cole crops, fiber crops, flowers, fruits, legume, oil crops, trees, tuber crops, vegetables, as well as other plants of the monocotyledonous, and dicotyledonous species. Preferably, crop seeds are coated which include but are not limited to bean, , corn, cotton, grasses, lettuce, peanut, pepper, potato, rapeseed, rice, rye, sorghum, soybean, sugar beet, sunflower, tobacco, and tomato seeds. Most preferably, barley or wheat (spring wheat or winter wheat) seeds are coated with the t compositions.

Also ed, in another aspect of the present invention, is a novel cereal plant created by artificially introducing a ial endophyte ofthe invention into a cereal plant that is free of endophytic microorganisms. In some ments ofthis aspect, the microbial endophyte introduced into the cereal plant may be an endophytic antagonist having suppressive activity against head blight disease or a causative agent of head blight disease.

Furthermore, the endophytic antagonist introduced into the cereal plant may be the fungal strain SGI—OlO-Hl l. A variety ofmethods previously found effective for the introduction of a microbial endophyte into cereal grass species are known in the art. Examples of such methods include those described in US. Pat. Appl. No. 20030195117A1, US. Pat. Appl. No. 20010032343Al, and US Pat. No. 7,084,331, among others. It will become apparent to those skilled in the art that many of the aforementioned methods can be useful for the making of a novel cereal plant of the invention.

After ial infection, it is preferred that DNA ofthe ed endophytic antagonist is ied by PCR and the antagonist is ed by ng out a homology search for the DNA amplified. Further it is preferred that a foreign gene that expresses an identifiable means is introduced into the above-mentioned endophytic antagonist, and the presence of the colonization of the above—mentioned endophytic antagonist infecting the plant is ed by the above-identifiable means using the foreign gene.

Preparing the trol compositions according to the present invention Cultures ofthe ial antagonists may be prepared for use in the biocontrol compositions ofthe invention using standard static drying and liquid fermentation techniques known in the art. Growth is commonly effected in a bioreactor.

A bioreactor refers to any device or system that supports a biologically active environment. As described herein a bioreactor is a vessel in which microorganisms including the ial antagonists of the invention can be grown. A bioreactor may be any appropriate shape or size for growing the microorganisms. A bioreactor may range in size and scale from 10 mL to liter’s to cubic meters and may be made of stainless steel or any other appropriate material as known and used in the art. The bioreactor may be a batch type bioreactor, a fed batch type or a continuous-type bioreactor (e.g. , a continuous stirred reactor). For example, a bioreactor may be a chemostat as known and used in the art of iology for growing and ting bacteria. A bioreactor may be obtained from any commercial supplier (See also Bioreactor System Design, Asenjo & Merchuk, CRC Press, 1995).

For small scale operations, a batch bioreactor may be used, for example, to test and develop new processes, and for processes that cannot be converted to continuous operations.

Microorganisms grown in a ctor may be suspended or immobilized. Growth in the bioreactor is generally under aerobic conditions at suitable temperatures and pH for growth. For the organisms of the invention, cell growth can be ed at temperatures n 5 and 37°C, with the preferred temperature being in the range of 15 to 30°C, 15 to 28°C, 20 to 30°C, or 15 to 25°C. The pH ofthe nutrient medium can vary between 4.0 and 9.0, but the preferred operating range is usually slightly acidic to neutral at pH 4.0 to 7.0, or 4.5 to 6.5, or pH 5.0 to 6.0. Typically, maximal cell yield is obtained in 20-72 hours after inoculation.

Optimal conditions for the cultivation of antagonists of this invention will, of in the course, depend upon the particular strain. However, by virtue of the ions d selection s and general requirements of most microorganisms, a person of ordinary skill in the art would be able to determine essential nutrients and conditions. The microbial nists would typically be grown in aerobic liquid cultures on media which contain sources of carbon, nitrogen, and inorganic salts that can be lated by the microorganism and supportive of efficient cell growth. Preferred carbon sources are hexoses such as glucose, but other sources that are readily assimilated such as amino acids, may be substituted. Many nic and proteinaceous materials may be used as nitrogen sources in the growth process. Preferred nitrogen sources are amino acids and urea but others include gaseous ammonia, nic salts of nitrate and ammonium, vitamins, purines, dines, yeast extract, beef extract, proteose peptone, soybean meal, hydrolysates of casein, distiller's solubles, and the like. Among the inorganic minerals that can be incorporated into the nutrient medium are the customary salts capable of yielding calcium, zinc, iron, manganese, magnesium, , cobalt, potassium, sodium, molybdate, phosphate, sulfate, chloride, borate, and like ions. Without being limited thereto, use of potato dextrose liquid medium for fungal antagonists and R2A broth premix for ial strains is preferred.

Throughout this disclosure, various information sources are referred 'to and incorporated by reference. The information sources include, for example, scientific journal articles, patent documents, textbooks, and World Wide Web r—inactive page addresses.

The reference to such ation sources is solely for the purpose ofproviding an indication of the general state of the art at the time of filing. While the ts and teachings of each and every one ofthe information s can be relied on and used by one of skill in the art to make and use embodiments of the invention, any discussion and comment in a specific information source should in no way be considered as an admission that such comment was widely accepted as the general opinion in the field.

The discussion of the general methods given herein is intended for illustrative purposes only. Other ative methods and embodiments will be apparent to those of skill in the art upon review of this disclosure, and are to be included within the spirit and purview ofthis application.

It should also be understood that the following examples are offered to rate, but not limit, the invention.

EXAMPLES EXAMPLE 1: Discoveg of the antagonistic microorganisms capable of suppressing the development ofFusarz'um gramz'naerum and Gibberella zeae, causative agents of head blight disease.

This example describes a high-throughput process of collecting and screening candidate microorganisms that has been developed internally at Synthetic Genomics, Inc. to isolate strains of microorganisms having suppressive activity against causative agents ofhead blight disease, particularly against Fusarium graminearum. Novel ial nistic strains were isolated from plant tissue samples collected from several locations across the United States. The bacterial strains SGIC06, SGI—Ol4-G01, SGI-OlS-F03, and SGI— 015-H06 were isolated from plant root tissues collected from Eagle Peak Preserve near , CA. The fungal strain SGI-OlO-Hll and the ial strain SGI-OOS-GOS were isolated from stem tissues oftwo different plant samples collected from San Elijo Lagoon, Encinitas, CA.

The rganisms were isolated as follows. For bacterial strain ion, plant root tissues were sonicated and subjected to serial dilutions on 2><YT (yeast extract and tryptone) and N—free agar medium plates. Individual colonies were then selected based on morphological characteristics, and dually cultured in liquid broth medium. For fimgal isolation, plant tissue was first surface-sterilized by dipping in 70% l and briefly passing through a flame. The tissue was then dissected and placed on potato dextrose agar (PDA) medium, followed by incubation at room temperature. When mycelial growth was observed, a segment of mycelial growth was then transferred to another PDA plate. Strains of microorganisms were isolated, d and preserved at —80°C in 15% glycerol at for bacterial strains and on dried barley seed for fungal strains.

Strains ofmicroorganisms ed as described above were thereafter assayed for their ability to suppress mycelial growth ofE graminearum in an in vitro antagonism test, which was performed on agar plates containing potato dextrose agar (PDA) growth medium according to a high-throughput screening procedure described in US. Pat. Appl. No.

U820120107915A1, which is incorporated by reference , with minor ations.

Briefly, isolated strains oorganisms were grown on one-fifth strength Tryptic soy broth agar S) for 24 hours prior to use. Conidial inoculum of F. gmmz'nearum NRRL—S883 was ed by hyphal tipping an actively growing colony ofthe fungus and transferring the hyphal strands to PDA agar medium. After incubating the plates for 7 days at °C using a 12 h/day photoperiod, conidia were washed from PDA plates using a weak phosphate buffer % phosphate buffer, pH 7.2, with 0.019% MgClz). A suspension of conidia of F. graminearum in the weak phosphate buffer (1 X 105 conidia/m1) was then immediately spread over the agar surface, and the plates were then incubated at 25°C for 48- 72 hours. To initiate the antagonism test, cells of isolated microbial s were point- inoculated at equal distances inside the perimeter of the plate. After five days, the s were scored as antibiosis positive when a y clear area that lacked mycelial growth existed around the perimeter of the microbial colonies.

More than 4,000 microbial strains were isolated and assayed by the procedure bed above. e, six strains were found to significantly suppress the mycelial growth ofF. gramz‘nearum NRRL—5883 on PDA medium. These new microbial antagonists are fied as SGI—005-G08, SGI-OlO-Hl 1, 4-CO6, SGI—014-G01, SGI-OlS-F03, and SGI-Ol 5—H06.

Antagonism test was also performed for the new microbial antagonists for their ability to suppress the growth of fungal pathogen Gibberella zeae, which is a teleomorphic species ofFusarz'um graminearum. All procedures were identical to those described above, except that the pathogen strain tested in this assay was a pathogenic strain of the fungus Gibberella zeae. Of these six microbial antagonists, four strains O-Hl 1, SGI—014- C06, SGI—015-F03, and SGI—015-H06 were found to significantly suppress the mycelial growth of Gibberella zeae.

EXAMPLE 2: DNA extraction, Sequencing and Taxonomy Fungal Cell Lysis and Acquiring ITS-5.88 rDNA Sequence Information The fungal biomass was transferred to a 96—well PCR microplate containing 50 ul of a 2 X lysis buffer (100 mM Tris HCL, pH 8.0, 2 mM EDTA, pH 8.0, 1% SDS, 400 ug/ml Proteinase K). Lysis conditions were as follows: 55°C for 60 minutes, 94°C for 4 minutes.

An aliquot ofthe lysis product was used as the source oftemplate DNA for PCR amplification. The ITS-5.88 rDNA sequence was amplified via PCR using two primers SI (SEQ ID NO: 15) and ITS4 iled (SEQ ID NO: 16).

For amplification ofthe ITS-5.88 rDNA region, each PCR mixture was prepared in a 20-ul final volume reaction containing 4 ul from the flmgal lysis reaction, 0.2 uM of each primer (ITSl/ITS4), 6% Tween-20, and 10 ul of 2X ImmoMix (Bioline USA Inc, Taunton, MA). PCR ions were as follows: 94°C for 10 minutes; 94°C for 30 seconds, 52°C for seconds, 72°C for 75 seconds for 30 cycles; 72°C for 10 minutes. A 2-ul aliquot ofthe PCR product was run on a 1.0% agarose gel to confirm a single band ofthe expected size.

Positive bands were sent out-for Sanger sequencing in the d and reverse directions using M13 primers. Sequence of the 5.88 enic region (ITS) of the fungal strain SGI- l is provided in the Sequence Listing as SEQ ID NO: 11. Homology search for the determined nucleotide sequence ofthe 5.88 intergenic region (ITS) was conducted using the DDBJ/GenBank/EMBL database. Subsequently, the phylogenetic relationship of the nucleotide sequence of the 5.88 enic region (ITS) was analyzed among the isolated fungal antagonist SGI-OlO—Hll described herein, rganisms of the genera and species that t high sequence homologies to the isolated fungal antagonist SGI-OlO-Hl 1, and other wide ies of rganism genera and, species, using the ClustalW phylogenetic tree ng program. The fimgal strain SGI-OlO-Hll is considered to be related to the family of Mycosphaerellaceae based upon ~97% similarity of its ITS-5.88 rDNA sequence to those ofMycosphaerella punctiformz‘s (GenBank EU343182) and Ramularz'a pratensz's (GenBank EU019284), whose 5.8S ITS sequences show clear relatedness to Mycosphaerellaceae.

Bacterial Cell Lysis and Acquiring 16S rRNA Sequence Information A 20-ul aliquot of cell suspension was transferred to a 96-well PCR plate containing 20 pl of a 2)< lysis buffer (100 mM Tris HCL, pH 8.0, 2 mM EDTA, pH 8.0, 1% SDS, 400 ug/ml Proteinase K). Lysis conditions were as follows: 55°C for 30 minutes, 94°C for 4 minutes. An aliquot of the lysis product was used as the source of template DNA for PCR amplification. The 16S rRNA sequence was amplified via PCR using M13—27F (SEQ ID NO: 17) and 1492R M13-tailed (SEQ ID NO: 18) primers.

For amplification of 16S rRNA region, each PCR mixture was prepared in a 20—u1 final volume reaction containing 4 pl from the bacterial lysis reaction, 2 uM of each primer (27F/1492R), 6% Tween-20, and 10 ul of 2X ImmoMix (Bioline USA Inc, Taunton, MA).

PCR conditions were as s: 94°C for 10 minutes; 94°C for 30 seconds, 52°C for 30 seconds, 72°C for 75 seconds for 30 cycles; 72°C for 10 s. A 2-ul aliquot ofthe PCR product was run on a 1.0% agarose gel to confirm a single band ofthe expected size. Positive bands were sent out for Sanger sequencing in the forward and e directions using M13 primers. Sequences of the 16S rRNA ofthe bacterial strains SGIC06, SGI-OOS-GOS, SGI—014-G01, SGIF03, and SGI—OlS—H06 are approximately 1.4—Kb in length and are provided in the Sequence Listing as SEQ ID NOS: 1, 10, 12, 13, and 14 respectively.

Homology search for the determined nucleotide ce was conducted using the DDBJ/GenBank/EMBL database. Subsequently, the phylogenetic relationship of the tide sequence ofthe 16 rRNA genes was analyzed among the isolated bacterial antagonists described , bacteria of the genera and species that exhibit high sequence homologies to the isolated bacterial antagonists, and other wide ies of bacterial genera and species, using the ClustaIW phylogenetic tree building program. Sequence identity and rity were also determined using GenomeQuestTM software (Gene-1T, Worcester Mass.

USA). The sequence is result revealed that the bacterial isolate SGI—014—G01 can be considered to be related to the genus of Variovorax based upon ~99% rity of its 16S rRNA sequence to those of several Variovorax species, including Variovorax sp. R—21938 (GenBank AJ786799) and Variovoraxparadoxus (GenBank GU186109), whose 16S rRNA sequences show clear dness to Variovorax sp. Further, the sequence analysis result also revealed that the two bacterial isolates SGI-015—F03 and SGI—015-H06 can be considered to be related to the family ofBacillaceae, based upon >99% similarity of their tive 168 sequences to those of several Bacillus spp.

The sequence analysis result revealed that the two bacterial isolates SGI—014-C06 and SGIG08 can be considered to be related to the family of Microbacteriaceae based upon >99% similarity of their respective l6S sequences to those of several acz‘erz’um spp. Notably, the 1430-nt sequence ofthe 16S rRNA gene of SGIC06 (SEQ ID NO: 1) is identical to the 16S rRNA gene of a Microbacterium oxydcms strain DSM 20578 and several other Mcrobacterium sp. strains (e.g. , k sequences Y17227.1, FJ200406, EU086800, and EU714335) over its entire 1430-nt length.

WO 16361 In particular, among the thousands ofmicrobial strains that were isolated and tested for the ability to suppress Fusarium development in antagonism assays as described in Example 1, a total of —eight strains was subsequently identified as Microbacterz'um species based upon the sequence similarity of their 168 sequences to those n Microbacterium spp. However, Applicants have found that SGI—014-C06 and SGI—OSS-GOS were the only two Microbacterium strains that possessed suppressive activity against Fusarium graminearum. To date, as discussed above, several naturally occurring microbes have been reported as having antagonistic activity against head blight disease. However, there are no reports prior to the present invention that describe a microorganism of the genus cterium having such antagonistic ty. Further, prior to the present invention, the present inventors were not aware of any methods or processes of using a bacterial strain of the genus cterz'um as biocontrol agent in preventing, inhibiting or treating the development of a causative en ofhead blight e.

EXAMPLE 3: Sequence Analysis of Housekeeping Genes from the Isolate SGI—Ol4-C06 A phylogenetic study of several housekeeping genes from 27 species of the genus Microbacterz'um has been reported recently by Richert et al. (Syst. App]. Mcrobiol. 30: 102- 108, 2007). The study concluded that although the merits of the 16S rRNA sequence analysis for systematic taxonomy are unsurpassed, sole emphasis on a single mic parameter ' should not guide systematic conclusions. As disclosed above, the nucleotide sequence ofthe 16S rRNA gene of SGIC06 (SEQ ID NO: 1) is identical to the 16S rRNA gene of several Microbacterium sp. strains, including the nucleotide sequence of a 16S rRNA gene of a acterium oxydans strain DSM 20578 that was included in t et al. (2007) study (GenBank Accession Y17227.1). Applicants proceeded to m a phylogenetic analysis on four housekeeping genes ofthe isolate SGIC06, which are DNA gyrase subunit B (gyrB), RNA-polymerase subunit B (rpoB), recombinase A (recA), and polyphosphate kinase (ppk). Toward this end, the entire genome of the isolate SGI-014—C06 was shot—gun sequenced, assembled and ted by using procedures described in PCT Patent Application No. W02010115156A2. Genomic DNA was ed from a fresh culture of SGIC06. Cell pellet was used for high molecular weight DNA extraction using the U1traC1ean® Mega Soil DNA Isolation Kit (Cat. No 12900-10) from MO BIO Laboratories, Inc. according to the manufacture’s recommended protocol. The genomic DNA from SGI— 014-C06 was then prepared for shotgun 454-pyrosequencing. Genomic DNA (7.5ug) was used for library construction according to the ended protocol (454 Life Sciences) for single long reads. The sequences were generated by two GS FLX Titanium series sequencing 111113.

The sequences of four housekeeping genes gyrB, rpoB, recA, andppk ofthe isolate SGIC06 are provided in the Sequence Listing. Homology search for the determined nucleotide sequences was conducted using the DDBJ/GenBank/EMBL database. Sequence identity and similarity were also determined using GenomeQuestTM software (Gene-IT, Worcester Mass. USA). As discussed in details below, the result of sequence analysis ofthe housekeeping genes revealed that the isolate SGIC06 described in the t disclosure is a novel bacterial strain and can be considered to be related to the family of Microbacteriaceae.

The polynucleotide sequence ofthe gyrB gene of SGI-Ol4-C06 has the st sequence identity with a gyrB gene obacterium testaceum having GenBank accession number AP012052 (82.69% over a 936/2172 nucleotide alignment). When compared to the gyrB gene ofthe Microbacterium s strain DSM 20578 (GenBank AM181493; Richert et al., 2007), the sequence gies between the two genes were ~62% identical at the tide level and ~37% identical at the amino acid level.

The polynucleotide sequence of the rpoB gene of SGI—Ol4-CO6 has the greatest sequence identity with an rpoB gene ofMicrobacterium maritypz‘cum having GeneBank accession number AM181582 (96.98% over a 1093/3504 nucleotide alignment). When ed to the rpoB gene of the Microbacz‘erium s strain DSM 20578 (GenBank 83; Richert et al., 2007), the sequence homologies between the two genes were ~96% identical at the tide level and 99% identical at the amino acid level.

The polynucleotide sequence of the recA gene of 4—C06 has the greatest sequence identity with a recA gene ofMicrobacterium testaceum having GeneBank accession number AP012052 (85.45% over a 962/1188 nucleotide alignment). When compared to the recA gene ofthe Microbacterium oxydans strain DSM 20578 (GenBank AM181527; Richert et al., 2007), the sequence homologies between the two genes were ~92% identical at the nucleotide level and 100% identical at the amino acid level.

The polynucleotide sequence of theppk gene of SGI—Ol4-C06 has the greatest sequence identity with appk gene ofMicrobacterium luteolum having k accession number AM181554 (91.38% over a 1380/2175 nucleotide alignment). When compared to the ppk gene ofthe Microbacterium oxydans strain DSM 20578 (GenBank AM181556; Richert et al., 2007), the sequence homologies between the, two genes were ~91% identical at the tide level and ~98% identical at the amino acid level.

EXAMPLE 4: tion of wheat from Fusarium graminearum infection using the microbial antagonists Microbacterium Sp. [ERRL B-50470) Mycophaerella sp. (N BBL 50471 1, and Variovorax sp. [EBB]: B-50469) Microbial strains that were found positive for antibiosis in the antagonism screen bed in Example 1 were further ted through a plant-based bioassay in which cells of the microbial strains were applied directly onto seeds of a susceptible cereal ar, followed by inoculation with al spores of F. grarninearum. The ial strains were grown to a sufficient turbidity and diluted in water. Two gram ofwheat seeds of a susceptible wheat cultivar (Hank; WestBred LLC, n, MT) were sown in one-liter pots containing pasteurized soil medium. After sowing, 20 mL ofthe microbial culture dilution was transferred on top of sown seeds. The wheat seeds were allowed to germinate in greenhouse conditions under fluorescent lighting with a 14 hour photoperiod. At the onset of wheat flowering, the wheat heads were nged by spraying with F. graminearum NRRL 5883 conidial spores. al spores were ted from five day old PDA plates by pouring water with 0.01% Tween20 on plates and scraping spores into suspension. After spraying spores, wheat plants were transferred to a mist chamber with 100% humidity for three days to allow for infection. The treatments were: l. Untreated: no microbial or chemical fungicide treatment + Fusarium challenge. . 2. Non-infected: no microbial or chemical fungicide treatment, no ’um challenge. 3. SGI—Ol O-Hl 1: fimgal treatment + Fusarium challenge. 4. SGI—014-G01: bacterial treatment + Fusarz'um challenge. 5. SGI-Ol4-C06: bacterial treatment + Fusarium challenge.

Twenty days after infection, watering was stopped and wheat plants were d to dry for three weeks before harvesting. Individual wheat heads were ted and collected. Disease severity was determined for each head showing head blight disease symptoms. Disease severity was calculated as the number of diseased spikelets divided by the total number of ets per heads. The results (TABLE 2) revealed that wheat plants treated with each of the three microbial antagonists tested, SGI-014—C06 (NRRL B-50470), SGI—OlO-Hll (NRRL 50471), and SGI—014-G01 (NRRL B-50469), had significantly lower severity and nce rz'um graminearum infestation 'when compared to the “untreated” control growing in the same conditions (P<0.05).

The seeds from each head were harvested by hand and the seed mass was weighed.

The average seed weight per pot in each ofthe treatments was calculated. As reported in TABLE 3, each ofthe ial antagonists was found to significantly reduce yield loss caused by head blight infestation.

TABLE 2. Efficacy ofthe microbial antagonists in reducing head blight incidence in Wheat.

Percent severity (%) STDEV SGI—OlO—Hll 0.0219 0.0005 SGI—014-G01 0.0315 0.0001 SGI—014-C06 0.0225 0.0004 TABLE 3. Efficacy of the microbial antagonists in preserving seed yield in wheat plants having head blight disease. 0.8438i0.1633 0.0191 SGI—OlO-Hll 0.6200 i 0 1844 0.7025 SGI—Ol4-G01 0.7963 3: 0 2032 0.5277 SGI—014-C06 7 0.6744 :i: 0.0948 0.0551 EXAMPLE 5: Growth tion ofFusarz'um graminearum by microbial antagonists on Was.

The ial strains found to possess suppressive activity against F. graminearum in antagonism plate assay were investigated further for their ability to reduce the incidence of head blight in wheat. Seeds of a susceptible wheat cultivar (Hank; ed LLC, Bozeman, MT) were sown in l-Liter pots each ning pasteurized soil medium in 20 cm diameter plastic pots. Microbial cell suspensions were prepared as follows.2YT, or similar growth medium, broth cultures were inoculated from isolate glycerol stocks or streak plates.

Typically, cultures were initiated 48 - 72 hours prior to use in the growth chamber, greenhouse or field allowing the culture to reach late ntial phase. Isolates that have longer doubling times were initiated further in advance. Cultures were incubated at 30°C on a rotary shaker at 200 rpm. After growth, the cells were pelleted at 10,000 x g for 15 min at 4°C and re-suspended in 10 mM MgSO4 buffer (pH 7.0). Cell densities were normalized for each isolate on a CFU/mL basis (cell performing units per milliliter). Typically, ~109 CFU/ mL suspensions were prepared for each isolate and transported to the ation site on ice.

Inoculations were performed by diluting these cell suspensions 1/20 in irrigation water to a final density 5 X 107 CFU/mL. For 1-L pot , 20 mL of this dilute cell suspension was buted evenly over the e of each replicate pot. For micro-trial pots, cell suspensions were not diluted and 1 mL of each 109 CFU/mL sion was pipetted directly onto the surface of each replicate pot as soil drench on top of the submerged seeds. The seeds and plants were maintained in a greenhouse at room temperature with a 14 h photoperiod of light.

Mycelium of the Fusarium gramz’nearum strain NRRL-5883 was grown on PDA plates for 5 days under constant light. Hyphae and conidia were harvested by g a few mL of e water (0.01% Tween 20) on the plates and scraping the agar surface with a sterile spatula. The tration of spores in the inoculum was approximately 2><105 spores/mL, but hyphal fragment concentration was not determined.

Inoculation with F. graminearum began after shoot emerging from the seeds and continued every other evening for 10 days. The inoculum of F. gramz'nearum was applied with a sprayer at about 30 mL per pot. Immediately after each inoculation, the plants were misted with overhead mister. When chemical fungicide was used, the fungicide (Banner Maxx, Syngenta) was prepared by diluting 2 mL of fungicide stock in IL of distilled water, and was applied with a sprayer at about 30 mL per pot.

Head blight was evaluated by the severity ofFusarium infestation, as determined by the number ofFusarium colony-forming units per gram (CFU/g) ofplant tissues. All treatments were performed in four blocks with four ate pots. The treatments were: 1. Untreated: no microbial or chemical fungicide treatment, + Fusarz'um challenge. 2. Non-infected: no microbial or chemical fungicide treatment, no Fusarium challenge. 3. Fungicide: chemical fungicide treatment + Fusarium challenge. 4. SGI-OlO-Hl 1: fungal treatment + Fusarium challenge. 5. SGIG01: bacterial treatment + Fusarz’um challenge. 6. SGI-014—C06: bacterial treatment + Fusarium challenge.

The results, as ed in TABLE 4, revealed that all three microbial treatments ed a significant decrease in FHB severity as compared to the infected control. In particular, two microbial antagonists, 4-C06 and SGI—OlO-Hl 1, significantly reduced the infestation of Wheat plants by um gramz’nearum when compared to the untreated control grown in the same conditions. The tion of Wheat plants from 'um infestation effected by each of the two microorganisms SGI—014-C06 and O-Hll was statistically comparable to the protection effected by the commercial al fungicide.

When the microorganism SGIG01 was applied, the observed protection of wheat plants from Fusarz'um infestation was much less conspicuous and its effectiveness varied greatly among the replicate pots.

TABLE 4: Effect ofmicrobial antagonists on Fusarz'um ation.

Treatment CFU/g Untreated 487 :l: 600 Non-infected <.0001 Chemical fungicide 290 i 146 <.0001 SGI-OlO—Hll 671 i 450 0.0077 4-G01 1246 i 465 0.8387 SGI—014-C06 367 i 234 0.0001 EXAMPLE 6: Growth and stora e of the microbial anta onists Mycosphaerella sp.: Several methods were used to store the isolated fungus as a pure culture, one of which was the filter paper technique. The fungus was also allowed to grow on PDA, and then it was cut into small squares which were placed into vials containing % glycerol and stored at —70°C. The fungus was also stored at 4°C by a similar method, using distilled water rather than glycerol. However, one of the preferred methods of storage was on infested sterile barley seed at -80°C.

Bacillus sp., Microbacterz'um Sp. and Variovorax sp.: The isolated bacteria were stored as a pure culture. A bacterial colony was transferred to a vial containing R2A broth liquid medium (Tecknova) and allowed to grow at 30°C with shaking at 250 rpm for two days. The culture was then transferred into vials containing 15% glycerol and stored at — 80°C.

EXAMPLE 7: Spore tion and seed coating treatments Spore tion: In a typical spore production procedure, one liter of2XYT growth medium (16 g/L ne, 10 g/L yeast extract, 5 g/L NaCl) was ated with 5 mL starter e or petri dish scraping and incubated overnight at 30°C in a ng shaker that was set at 225 rpm. Bacterial cells were pelleted by centrifugation, and washed 1x with PBS buffer (8 g/L NaCl; 0.2 g/L KCI; 1.44 g/L NazHPO4; 0.24g of KH2P04; pH 7.4). Cells were resuspended in CDSM medium (Hageman et al., J Bacteriol., 43 8-441, 1984), and were grown for four additional nights at 30°C in the rotating shaker. Sporulation in the bacterial culture was monitored daily by using phase contrast microscope until the entire culture was virtually made up of free-floating spores. The tion time typically took less. than four days or more than six days depending on the s. Under phase contrast microscope, endospores were detected within cells as bright white oblate spheroids. Bacterial spores were pelleted by centrifugation at 10,000 X g for 15 minutes, washed two times with sterile dH20 and, if necessary, concentrated down to 50 mL and could either be used immediately or refrigerated for later use. Spore concentration was measured at 0D600. This procedure typically produced at least 2000 OD's of bacterial spores.

In particular, several bacterial strains of the present invention, e.g. Bacillus sp.

SGIF03, could conveniently produce large amounts of spores after 4 days of incubation with a simple inoculation of a large overnight starter culture (~15 ml.) in to 1 liter of 2>< SG growth medium, followed by a 4-day incubation at appropriate temperature in a rotating shaker set at 225 rpm. The recipe of the 2x SG growth medium was as follows: 16 g/L Nutrient Broth; 0.25 g/L MgSO4; 2.0 g/L KCl; 0.15 g/L 2H20; 0.025 g/L MnC12.2H20; 0.28 mg/L FeSO4-7H20; 1.0 g/L Dextrose.

Seed coating treatments ofwheat and corn seeds: Small-scale seed treatment experiments were conducted by ing a procedure described in Sudisha et al. , 2009 with minor ations. Typically, a biopolymer stock solution was made by adding 6 gram of gum Arabic powder (MP Biomedical) to 36 mL water in a 50 mL Falcon tube, which was subsequently mixed to neity by using a wheel mixer. A stir plate was used for mixing when larger quantities of coated seeds were .

When vegetative cells were used, turbid cultures of actively growing microbial cells were washed with PBS and adjusted to an OD600 of ~5.0. Alternatively, microbial spore suspensions were prepared as described above. Bacterial spores and/or vegetative cells were thoroughly resuspended in ~32 mL gum Arabic biopolymer stock solution prepared as described above, and resulting suspension was mixed thoroughly in a 1 L bottle.

Approximately 400 g of seeds (either wheat or corn) were added to the bottle and vigorously shaken or vortexed to ensure a m distribution ofthe ll sion. Coated seeds were then spread across a sterile plastic weigh boats to dry in a laminar flow hood until no longer tacky, generally 3-6 hours with periodic . In some instances, seeds coated with spores could be dried overnight. However, seeds coated with vegetative cultures were typically stored away before they completely desiccated. Viability test performed WO 16361 ically on the microbes used in seed coating formulation showed that the es remained viable for at least three months. Germination rate ofthe coated seeds was determined to be essentially identical to control uncoated seeds.

EXAMPLE 8: Effect of microbial seed treatments on the development of Fusarium head blight in wheat Seeds of a FHB-susceptible wheat cultivar (RB07) were coated with each ofthe microorganisms SGI-014—C06; SGI—OlS-F03, and SGI-OlS-HOG, in accordance with the procedure described in Example 8. Coated seeds were subsequently sown in one-liter pots containing pasteurized soil medium [(Metromix—Mix 200; -Sierra Horticultural Products, ille, OH; and 3 gram of fertilizer 14—14—14 (N-P—K)]. Pots were seeded at 7-8 plants per pot and subsequently thinned to 5 plants per pot at the 3—leaf stage. Pots were ed in randomized blocks with 5 pots per treatments. The coated seeds were then allowed to germinate in ouse conditions under fluorescent ng with 16 hours of lighting per day. At the onset of wheat flowering (anthesis), the wheat heads were challenged by spraying with F. graminearum NRRL 5883 macroconidial spores at a concentration of 100,000 spores/mL. After spraying spores, wheat plants were transferred to a dew chamber with 100% ty for three days to allow for infection. The treatments were: 1. Untreated: no microbial or chemical fungicide treatment + Fusarz'um challenge. 2. SGIC06: bacterial seed treatment + Fusarium challenge. 3. SGI—Ol 5-F03: bacterial seed treatment + Fusarium challenge. 4. SGI-OlS-H06: bacterial seed treatment + Fusarium challenge.

Severity of head blight disease was ed visually at ten days and twenty-one days after infection. Disease severity was determined for each spike showing head blight disease symptoms, and calculated as the percentage of matic spikelets; i. e. the number of spikelets displaying FHB symptoms divided by the total number of spikelets. The results (TABLE 5) revealed that wheat seeds coated with each of the three ial antagonists tested, SGIC06; SGI-OlS—FO3, and SGI-OlS-H06, had significantly lower severity of Fusarz'um graminearum infestation when compared to the ated” control growing in the same conditions (P<0.05).

Table 5: Development of head blight symptoms in wheat following seed treatments with antagonistic microorganisms _10 days post-infection SGI-014—C06 2.08 S-F03 23.12 E 9: Biocontrol ofhead-blight disease of wheat in greenhouse trials Each of the microorganisms SGICO6; SGI—OlS-FO3, and SGI—OlS-H06 were further tested at larger-scale greenhouse trials. The trials were conducted in a “plant growth containment room” (PGCR) located at Synthetic Genomics, Inc., and included the following control treatments: infected control, fected control, as well as four commercial fungicide benchmarks. The benchmarking chemical fungicides were Banner MAXX® (Syngenta) at a concentration of 2 ml/L and Prosaro® (Bayer CropScience) at a concentration of 3 ml/L, each applied as a foliar spray, following manufacturers’ recommendation. The benchmarking biological firngicides were Actinovate® al Industries) and RhizoVital® (ABiTEP GmbH), each applied as a soil drench as per instruction s recommended by manufacturers.

The microbial treatments were prepared and applied either as a soil drench or as a seed coat. When ial treatments were applied as a soil drench, cell suspensions of each of the microorganisms were individually applied to the seeds before covering the seeds with more growth medium. For this purpose, freshly prepared cultures were pelleted to remove growth media and resuspended in 10 mM ium sulfate (MgSO4) buffer. Cell suspensions were then added by ing and buting 20 milliliters evenly across the seeds at a total cell number of ~1 09 cells per pot. When microbial treatments were applied as a seed coating, microorganism cells/spores were essentially ed as described in Example 8 above. Coated seeds were produced by incorporating cell suspensions with a gum Arabic solution to form a tacky coating mixture that was subsequently applied to the seeds. The microbial seed coating was then allowed to dry to form a hard but water soluble biopolymer entrapping the rganism cells/spores. The goal was to reach a cell titer in the range of at least 106 — 107 viable cells/seed. In this greenhouse trial ment, the seed coatings were prepared the day prior to the sowing ofthe seeds.

Each treatment had four replicates that were positioned in a Randomized Complete Block Design (RCBD) on three leveled racks arranged in two rows along the sides of a growth room. The racks were ed with fluorescent lights (Agrosun, 5850K) that generated on average 800 umole of light intensity as measured at the pot surface. Each replicate consisted of four l-liter pots contained in a half flat. The pots were filled with a synthetic growth medium consisted of a 3:1 ratio (V/v) of Arabidopsis Growth Medium AIS (Lehle Seeds) to sand (Washed Coarse). In each pot, two grams of spring wheat seeds (Hank, WestBred) were scattered on the surface ofthe growth medium. The pots were bottom watered by keeping ~2 cm water at their base and ed for germination and growth.

At ng stage (Feekes ) the wheat heads were infected by spraying with a Fusarium gramz'nearum conidial spore suspension. For this greenhouse trial experiment, fungal spores were prepared by plating homogenized mycelia ofF. gramz'nearum strain NRRL 5883 onto large PDA plates, followed by five-day incubation under constant light at room temperature. The fungal spores were then harvested by flooding the plates with magnesium sulfate buffer (10 mM MgSO4; 0.01% Tween 20) and gently scraping the agar e with a sterile spatula. The spore concentration was adjusted and approximately 50,000 conidial spores were applied per head with a pressurized hand sprayer. The growth room ve humidity was increased to 90% for three days allowing for ion before again being normalized. After wheat seeds had set, watering was d and the wheat heads were allowed to dry completely. Individual wheat heads were ted and collected.

Disease severity was determined for each wheat head showing head blight disease symptoms.

Disease severity was calculated as the number of diseased spikelets divided by the total number of spikelets per head. The results (TABLE 6) revealed that wheat treated with each ofthe three microbial antagonists tested, SGIC06; SGI-OlS-F03, and SGI-OlS-HO6, had significantly lower severity and incidence ofFusarz'um graminearum infestation when compared to the “untreated” l growing in the same conditions (P<0.05).

TABLE 6. Efficacy of the microbial antagonists in reducing head blight incidence in wheat.

Infection severity (%) Infection protection% ——-u- SGI-OlS-H06 26.6 i 7.4 34.00 To determine seed yield, wheat heads were individually threshed by hand, the seeds collected and d of chaff and other detritus, counted, and d. The total seed yield was determined as the total seed mass produced by 16 pots of each ofthe treatments.

As reported in TABLE 7 each ofthe three microbial nists tested was found to significantly protect the seed yield; 1'. e. the yield loss caused by head blight infestation was significantly reduced.

TABLE 7. Efficacy ofthe microbial antagonists in preserving seed yield in wheat plants having head blight disease. ——-a- SGI—OlS-FO3 S-H06 Thus, treatments of wheat plants with each of the tested microorganisms significantly preserved the wheat plants t yield loss caused by Fusarz‘um infestation. In particular, wheat plants treated with either SGI—014-C06 or SGI-OlS-F03 showed a significant improvement in total seed yield; z'.e. 110% and 120% respectively, when compared to non-infected control. In contrast, the benchmarking fungicides Actinovate®, RhizoVital®, and Prosario® did not appear to significantly protect the treated Wheat plants from yield loss caused by Fusarium head blight infestation.

E 10: Biocontrol ofwheat scab under field conditions.

Microorganism antagonists that were found having suppressive activity t the en F. graminearum and head blight disease in in vitro antagonism assays and greenhouse studies, as described in Examples 4-9, are further evaluated in a series field experiments at ent geographical locations throughout the United States. Some experiments are carried out in agricultural areas which are used for iological control of wheat scab, where natural disease infection occurs. The wheat variety used in the test is a F. graminearum susceptible variety. Generally, Wheat plants with each treatment were sown in plots of 12 rows, about 3-meters long. The space between rows is about 20 cm. Treated plots in each ment are arranged in a randomized block design. The efficacy of various wettable powder itions containing the microbial antagonists ofthe present invention in reducing head blight severity and incidence are also assessed. In these experiments, the microbial antagonists are evaluated either individually or in combination.

In these tests, microorganism antagonists are evaluated in various seed-coating treatments and/or field tests where microbial suspensions are applied directly onto ng wheat heads. In each ofthe experiments, the microorganisms are assessed in replicated plots.

Antagonist, en, and plant production methods as described in Examples 4 -9 can be used in these experiments.

Seed-coating treatment — In on to the seed coating method described in Example 7 above, a variety oftechniques and seed-coating ations known in the art can be deployed for the treatment ofwheat seeds with microbial antagonists, such as those described usly by Fernando et al., 2002; Bello et al. , 2002; and Kim et al, 1997. In general, wheat seeds are pre-moistened and stored at room temperature to promote ation. ated seeds can be then immersed into microbial sions before seeding. The pathogen spores can be inoculated either before or after bacterial inoculation.

Antagonist, pathogen, and plant production methods as described in Examples 4 and 5 can be used for these seed treatments.

Disease severity and yield assessment — The antagonists are subjected to further evaluation in greenhouse and field evaluation for their suppressive activity against head blight disease on wheat and barley at ng stages, by using a variety of methods and procedures, typically those described in, e.g., Schisler, Plant Disease, Vol 86(12), 1350—1356, 2002; Schisler et al. Biological Control, 39:497—506, 2006; and Khan and Doohan, Biological l, 48:42-47, 2009. In one of such experiments, inoculums of G. zeae are prepared on sterile, yellow dent corn as described by Khan et al. (Biological Control, 29:245-255, 2004).

Fully colonized lipid cultures ur culture) of microbial strains are diluted to one-quarter strength with ate buffer. Final CFU per mL counts for antagonist treatments are between 1 X 109 CFU/mL and 6 X 109 CFU/mL. Treatment suspensions are then applied to flowering wheat heads using a C02 backpack sprayer. Treatments are typically d after sunset to minimize potential UV degradation of antagonist cells. There are 5 replicates per treatment which are arranged in a randomized block . The primary control treatment consists of plants treated with a buffer solution. A second control consists of untreated plants. Field assessments of head blight e and incidence are made by evaluating 60 heads per ate (i. e. 300 heads/treatment) When plant development reaches n mid- milk and soft dough development. Wheat heads are harvested by hand and threshed using an Almaco single plant and head thresher (Almaco, IA) when grains reach full maturity. Grain samples obtained fiom each replicate row are evaluated for lOO—kernel weight. Disease severity, incidence, and 100-kernel weight data are ed using one-way analysis of variance (ANOVA).

The compositions containing the microbial antagonists ofthe invention, either individually or in combination, are found to significantly diminish the disease incidence.

These results show that the biological control measures using these active microbial agents can play an important role in the management of scab on cereal plants such as wheat .

EXAMPLE 11: Development ofnon-naturally occurring cultivars and breeding program Endophytic microorganisms ofthe present ion are introduced into crop plants, including s, of varying genotypes and geographic origin, lacking such endophytic rganisms, to create plant-endophyte combinations with improved agronomic characteristics, using procedures analogous to those known in the art, including those described in US. Pat. Appl. No. 20030195117A1; US. Pat. Appl. No. 20010032343A1; and U.S. Pat. No. 7,084,331, among others. Thus, given synthetic plant- endophyte combinations may be created and selected in a breeding/cultivar pment program based on their ability to form and maintain a mutualistic combination that results in ' an agronomic benefit. Rating of agronomic characteristics of the combination may also be utilized in such a breeding m. These characteristics may include, without limitation, drought tolerance, biomass accumulation, resistance to insect infestation, palatability to livestock (e.g. herbivores), ease of reproduction, and seed yield, among others. Such combinations may differ in levels of accumulation of microbial metabolites that are toxic to pests and weeds, including ergot alkaloid levels, loline levels, peramine levels, or lolitrem levels, while displaying desired agronomic characteristics of crop plants, including resistance to insect g or infestation, resistance to abiotic stress, palatability to ock, biomass accumulation, ease of reproduction, and seed yield, among other traits.

A number ofembodiments of the ion have been described. Nevertheless, it will be understood that elements of the embodiments described herein can be combined to make additional embodiments and various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments, alternatives and equivalents are within the scope of the invention as described and claimed herein.

Headings within the application are solely for the convenience of the , and do not limit in any way the scope of the ion or its embodiments.

All publications and patent applications ned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically can individually indicated to be incorporated by reference. 5555 BUDAPEST TREATY ON THE INTERNATIONAL RECOGNITION OF THE DEPOSIT OF MICROORGANISMS FOR THE PURPOSE OF PATENT PROCEDURES INTERNATIONAL FORM TO RECEIPT IN THE CASE OF AN ORIGINAL DEPOSIT Patent Dept. issued pursuant to Rule 7.1 by the Synthetic Genomics INTERNATIONAL DEPOSITARY AUTHORITY 1 1149 North Torrey Pines Rd. identified at the bottom of this page Lajolla, CA 92037 NANIE AND ADDRESS OF DEPOSITOR I. FICATION OF THE MICROORGANISM Identification reference given by the DEPOSITOR: Accessmn number given by the [wimp/Jamfla 1}). INTERNATIONAL DEPOSITARY ITY: 010 H11 NRRL 50471 II. IFIC DESCRIPTION AND/OR PROPOSED TAXONOMIC DESIGNATION The microorganism identified under I. above was accompanied by: E] 1a scientific description Eda proposed taxonomic designation III RECEIPT AND ACCEPTANCE This International Depositary Authority accepts the microorganism identified under I above, which was received byit February 18, 2011(date of the original deposit)z IV. RECEIPT OF RE. UEST FOR CONVERSION The microorganism fied under I above, was received by this International Depositary ity on_— (date of the original deposit) and a request to t the original deposit to a deposit under the Budapest——Treaty was received byit on (date of receipt of request for conversion).

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SEQUENCE LISTING <110> SYNTHETIC GENOMICS, INC.

GRANDLIC, CHRISTOPHER J.

GREEN, WAYNE A.

KEROVUO, JANNE S.

MCCANN, RYAN T. <120> ITIONS AND METHODS FOR CONTROLLING HEAD BLIGHT DISEASE <130> SGI1520-1WO <150> US 61/511,467 <151> 201125 <160> 18 <170> PatentIn n 3.5 <210> 1 <211> 1430 <212> DNA <213> Microbacterium sp. <220> <221> misc_feature <223> SGI bacterial isolate 4-C06 <220> <221> misc_feature <223> encodes a 16S ribosomal RNA <400> 1 acgctggcgg cgtgcttaac acatgcaagt cgaacggtga acacggagct tgctctgtgg gatcagtggc gaacgggtga gtaacacgtg agcaacctgc ccctgactct gggataagcg ctggaaacgg cgtctaatac tggatatgtg acgtgaccgc atggtctgcg tctggaaaga atttcggttg ggct cgcggcctat cagcttgttg gtgaggtaat ggctcaccaa ggcgtcgacg ggtagccggc ctgagagggt gaccggccac actgggactg agacacggcc cagactccta cgggaggcag cagtggggaa acaa tgggcgcaag cctgatgcag caacgccgcg tgagggacga cggccttcgg gttgtaaacc tcttttagca gggaagaagc gaaagtgacg gtacctgcag cgcc ggctaactac gtgccagcag ccgcggtaat ggcg caagcgttat ccggaattat tgggcgtaaa gagctcgtag tgtc gcgtctgctg tgaaatccgg aggctcaacc tccggcctgc agtgggtacg ggcagactag gtag gggagattgg aattcctggt gtagcggtgg aatgcgcaga tatcaggagg aacaccgatg gcag atctctgggc cgtaactgac gctgaggagc gaaagggtgg ggagcaaaca ggcttagata ccctggtagt ccaccccgta aacgttggga actagttgtg gggtccattc cacggattcc gtgacgcagc taacgcatta agttccccgc ctggggagta cggccgcaag gctaaaactc aaaggaattg acggggaccc gcacaagcgg cggagcatgc ggattaattc acgc gaagaacctt accaaggctt gacatatacg ggcc ggtc aactctttgg acactcgtaa acaggtggtg catggttgtc gtcagctcgt 1020 gtcgtgagat gttgggttaa gtcccgcaac gagcgcaacc ctcgttctat gttgccagca 1080 cgtaatggtg ggaactcatg tgcc ggggtcaact aagg tggggatgac 1140 gtcaaatcat catgcccctt atgtcttggg cttcacgcat gctacaatgg ccggtacaaa 1200 gggctgcaat accgcgaggt ggagcgaatc ccaaaaagcc ggtcccagtt cggattgagg 1260 tctgcaactc gacctcatga agtcggagtc gctagtaatc cagc aacgctgcgg 1320 tgaatacgtt cccgggtctt gtacacaccg aagt catgaaagtc ggtaacacct 1380 gaagccggtg gcctaaccct tgtggaggga gccgtcgaag gtgggatcgg 1430 <210> 2 <211> 2172 <212> DNA <213> Microbacterium sp. <220> <221> misc_feature <223> SGI bacterial isolate 4-C06 <220> <221> misc_feature <223> SGI Gene ID SG1MICG850682 <220> <221> misc_feature <223> encodes the e sequence at SED ID NO 3 <400> 2 gtgtctgcgg ccggagagcg gctgtcgaga atagactcgg agattgtgaa tgccgagtat tccgcccatc atctccaggt gcttgagggg ctcgaagctg tccgcaagcg cccgggcatg tacatcgggt cgaacggctc gccgggcctc atgcactgcc tttgggagat catcgacaac tctgtcgacg aggcggtggc aggcaacggc acgaagatcg acatcatcct gcactccgac ggcagcgtcg aggtgcacga tcgc ggcatccctg tcgacgtcga gccgcgcacc ggtctcaccg gtgtcgaggt cgtctacacc aagctgcacg ccggaggaaa gttcggcggc ggctcgtacg cggcatccgg tggactgcac ggtgtcggcg cctccgtcgt gaacgcgctc tccgagcgcc tcgacgtcga ggtcgaccgc gggggcaaga cctacgcgat gtcgttccat cgcggtgagc ccggcatctt cacggactcc ggcgagaagc ggccggacgc cccgttcacg ccgttcgagg agaacagcga gctgcgcgtc atcggcaagg cgccgcgtgg cgtgaccggc acccgggtgc gctactgggc cgaccggcag atcttcacca cggc gttccagctc tcggagctcg gcgc gacg gcgttcctcg ttcccggtct cgagatcgtc gtcaaggacg cacgggcggc ggtc atccccgtcc ccga cggcgagacg accgtcgtcg atga gacctcgtac ctctacgagg gcggcatctc cgagttcgtc gagtacctcg ccatcgaccc tcccgtgacc gacacctggc gtatccaggg cgagggcatg ttcaaggaga ccgt cctgcaggca gacggccaca tggtcgccac ggag cgtgtgtgcg ccgtcgacat cgcgctgcgc tgggggaccg gctacgacac ccgtgtgcgc 1020 tccttcgtga acatcatcgc gacgcccaag ggcggaaccc accagcaggg cttcgagcag 1080 ctga aggtgctgcg ctcccaggtc gagcagaacg cccgccgtct gaaggtcggc 1140 aacgacaagc tggagaagga cgacgtcctc gccggcctca ccgccgtgct cacggtcaac 1200 gtgccggagc cgcagttcga gggccagacc aaggaagtgc tcggcacccc cgcggtgcgg 1260 cagatcgtgg cgcaggtgat ccgtaaggat ctggcgcagc gctc gaccaagcgc 1320 gacgacaaga accaggccac acagctgctc gacaagatcg tctccgagat gaaggcccgt 1380 gcgc gcgcccacaa ggagacgcag cgccgcaaga acgcgctgga gtcgtcgacg 1440 ctgccgacca agctcgtcga ctgccgcacg gtcg gcga gctcttcatc 1500 gtggagggcg actcggctct cggcaccgcc aagaacgcgc gcaacagcga gttccaggcg 1560 ctgctcccga tccgagggaa gatcctcaac aagg cctctgtcgg cgacatgctg 1620 tcgaacaccg agtgcgcgtc gatcatccag gtgatcggcg ccggatccgg acgcaccttc 1680 gacatcgatg cggcgcgcta cggcaaggtg atcctgatga gcgacgccga tgtcgacggc 1740 gcgcacatcc gtaccctgct gctcacgctg ttcttccgct acatgcgacc gctgatcgag 1800 cacgggcgtg tgttcgccgc ggtgccgccg ttgcaccggg tgatcgtgat gaacccgggg 1860 tccaagccga cgat ctacacctac agcgagcagg acgc gctgctggcg 1920 aagctccgca aggccggcaa gcgctggcac gagccgatcc agcgctacaa gggtctcggt 1980 gagatggacg cggaacagct cgcgaacacc accatggacc gctccggccg tctgctgcgc 2040 cgtgtgcgca tggaagacgc cgaggccgcc ggtcgcgtgt tcgagctgct gatgggcaac 2100 gaggtcgcgc cgcgccgcga gttcatcatc gactcctccg accggttgtc gtcc 2160 atcgacgcct ga 2172 <210> 3 <211> 723 <212> PRT <213> Microbacterium sp. <220> <221> misc_feature <223> SGI bacterial isolate SGIC06 <220> <221> misc_feature <223> SGI peptide ID SG1MICT850682 <220> <221> misc_feature <223> gyrase subunit B <400> 3 Met Ser Ala Ala Gly Glu Arg Leu Ser Arg Ile Asp Ser Glu Ile Val 1 5 10 15 Asn Ala Glu Tyr Ser Ala His His Leu Gln Val Leu Glu Gly Leu Glu 25 30 Ala Val Arg Lys Arg Pro Gly Met Tyr Ile Gly Ser Asn Gly Ser Pro 40 45 Gly Leu Met His Cys Leu Trp Glu Ile Ile Asp Asn Ser Val Asp Glu 50 55 60 Ala Val Ala Gly Asn Gly Thr Lys Ile Asp Ile Ile Leu His Ser Asp 65 70 75 80 Gly Ser Val Glu Val His Asp Arg Gly Arg Gly Ile Pro Val Asp Val 85 90 95 Glu Pro Arg Thr Gly Leu Thr Gly Val Glu Val Val Tyr Thr Lys Leu 100 105 110 His Ala Gly Gly Lys Phe Gly Gly Gly Ser Tyr Ala Ala Ser Gly Gly 115 120 125 Leu His Gly Val Gly Ala Ser Val Val Asn Ala Leu Ser Glu Arg Leu 130 135 140 Asp Val Glu Val Asp Arg Gly Gly Lys Thr Tyr Ala Met Ser Phe His 145 150 155 160 Arg Gly Glu Pro Gly Ile Phe Thr Asp Ser Gly Glu Lys Arg Pro Asp 165 170 175 Ala Pro Phe Thr Pro Phe Glu Glu Asn Ser Glu Leu Arg Val Ile Gly 180 185 190 Lys Ala Pro Arg Gly Val Thr Gly Thr Arg Val Arg Tyr Trp Ala Asp 195 200 205 Arg Gln Ile Phe Thr Lys Asp Ala Ala Phe Gln Leu Ser Glu Leu Glu 210 215 220 Thr Arg Ala Arg Gln Thr Ala Phe Leu Val Pro Gly Leu Glu Ile Val 225 230 235 240 Val Lys Asp Ala Arg Ala Ala Gly Gln Val Ile Pro Val Pro Asp Ser 245 250 255 Asp Gly Glu Thr Thr Val Val Gly Thr Asp Glu Thr Ser Tyr Leu Tyr 260 265 270 Glu Gly Gly Ile Ser Glu Phe Val Glu Tyr Leu Ala Ile Asp Pro Pro 275 280 285 Val Thr Asp Thr Trp Arg Ile Gln Gly Glu Gly Met Phe Lys Glu Thr 290 295 300 Val Pro Val Leu Gln Ala Asp Gly His Met Val Ala Thr Glu Val Glu 305 310 315 320 Arg Val Cys Ala Val Asp Ile Ala Leu Arg Trp Gly Thr Gly Tyr Asp 325 330 335 Thr Arg Val Arg Ser Phe Val Asn Ile Ile Ala Thr Pro Lys Gly Gly 340 345 350 Thr His Gln Gln Gly Phe Glu Gln Glu Leu Leu Lys Val Leu Arg Ser 355 360 365 Gln Val Glu Gln Asn Ala Arg Arg Leu Lys Val Gly Asn Asp Lys Leu 370 375 380 Glu Lys Asp Asp Val Leu Ala Gly Leu Thr Ala Val Leu Thr Val Asn 385 390 395 400 Val Pro Glu Pro Gln Phe Glu Gly Gln Thr Lys Glu Val Leu Gly Thr 405 410 415 Pro Ala Val Arg Gln Ile Val Ala Gln Val Ile Arg Lys Asp Leu Ala 420 425 430 Gln Arg Phe Ser Ser Thr Lys Arg Asp Asp Lys Asn Gln Ala Thr Gln 435 440 445 Leu Leu Asp Lys Ile Val Ser Glu Met Lys Ala Arg Val Ser Ala Arg 450 455 460 Ala His Lys Glu Thr Gln Arg Arg Lys Asn Ala Leu Glu Ser Ser Thr 465 470 475 480 Leu Pro Thr Lys Leu Val Asp Cys Arg Thr Asn Glu Val Glu Arg Ser 485 490 495 Glu Leu Phe Ile Val Glu Gly Asp Ser Ala Leu Gly Thr Ala Lys Asn 500 505 510 Ala Arg Asn Ser Glu Phe Gln Ala Leu Leu Pro Ile Arg Gly Lys Ile 515 520 525 Leu Asn Val Gln Lys Ala Ser Val Gly Asp Met Leu Ser Asn Thr Glu 530 535 540 Cys Ala Ser Ile Ile Gln Val Ile Gly Ala Gly Ser Gly Arg Thr Phe 545 550 555 560 Asp Ile Asp Ala Ala Arg Tyr Gly Lys Val Ile Leu Met Ser Asp Ala 565 570 575 Asp Val Asp Gly Ala His Ile Arg Thr Leu Leu Leu Thr Leu Phe Phe 580 585 590 Arg Tyr Met Arg Pro Leu Ile Glu His Gly Arg Val Phe Ala Ala Val 595 600 605 Pro Pro Leu His Arg Val Ile Val Met Asn Pro Gly Ser Lys Pro Asn 610 615 620 Glu Thr Ile Tyr Thr Tyr Ser Glu Gln Glu Met His Ala Leu Leu Ala 625 630 635 640 Lys Leu Arg Lys Ala Gly Lys Arg Trp His Glu Pro Ile Gln Arg Tyr 645 650 655 Lys Gly Leu Gly Glu Met Asp Ala Glu Gln Leu Ala Asn Thr Thr Met 660 665 670 Asp Arg Ser Gly Arg Leu Leu Arg Arg Val Arg Met Glu Asp Ala Glu 675 680 685 Ala Ala Gly Arg Val Phe Glu Leu Leu Met Gly Asn Glu Val Ala Pro 690 695 700 Arg Arg Glu Phe Ile Ile Asp Ser Ser Asp Arg Leu Ser Arg Glu Ser 705 710 715 720 Ile Asp Ala <210> 4 <211> 3504 <212> DNA <213> Microbacterium sp. <220> <221> misc_feature <223> SGI ial isolate SGIC06 <220> <221> misc_feature <223> SGI Gene ID SG1MICG850290 <220> <221> eature <223> encodes the peptide sequence at SEQ ID NO 5 <400> 4 ttggctgctg acgc atccacatcc accaccacca agaacggacg cggagcttcc cgtctttcgt tcgccaagat ctccgacacg ctgacggtcc ctgaccttct gcag accgaatcct tcggttggct ggtcggcaac tgga gcgt ggccgaggcc aagaagcagg ggcgcaccga cgtcaacgag aacagcggtc tgggcgagat cttcgaggag atctctccga tcgaggacct gacg atgcagctgt cgttcacgaa cccctacctc gaga agtactccat cgaggagtgc aaggagcgtg gcaagaccta cgccgctccg ctgtacgtcg aggccgagtt catgaaccac ctcacgggtg agatcaagac ccagacggtc ttcatgggcg cgct gcagaccgac acgt tcatcatcaa cggctccgag cgcgtcgtcg agct ggtgcgttcg cccggtgtct acttcgacaa gacccccgac aagacgtccg acaaggacat cgtctcggca cgcgtcatcc cgagccgtgg tgcctggctc gagttcgaga tcgacaagcg cgaccaggtc ggcgtgcgcg tcgaccgcaa gcgcaagcag tcggtcacgg tcttcctcaa ggcgctgggc atgaccagcg aggagatcct cgccgagttc gccggctaca cctcgatcga ggagacgctc gcgaaggaca tcac gaaggaagat gcgctccgcg acatctaccg caagctccgt ccgggcgagc aggtcgccgc cgaggccgcc cgcgcgctcc tggacaactt ctacttcaac ccgaagcgct acgacctggc caaggtcggt cgctacaaga tcaaccacaa gctgggcctg gaccagccgc tgaactcgtc ggtcctgacc gtcgaagaca tcgtggccac gatcaagtac ctggtgcgtc tgcacgccgg caccgaggag 1020 accttcacgg gcatccgcgg tggtaagaag gccgagatcc gtctcgcgac cgacgacatc 1080 gacaacttcg gcaaccgtcg catccgcgcg gtcggcgagc tgatccagaa ccaggtccgc 1140 accggtctct tgga gcgcgtcgtc cgcgagcgca cgca ggacatcgag 1200 gcgatcacgc cgcagaccct gatcaacgtg cgccccgtcg tcgccgcgat caaggagttc 1260 ttcggaacgt cgcagctgtc gcagttcatg gaccagaaca acccgctcgc gggtctgacg 1320 aacaagcgtc gtctgtctgc gctcggcccc ggtggtctct cgcgagaccg cgccggcgtc 1380 gaggtccgtg acgtccaccc ctcgcactac ggccgcatgt gcccgatcga gactccggaa 1440 ggcccgaaca tgat cggtgctctc gcgaccttcg cgcgcatcaa cgga 1500 ttcatcgaga ccccgtaccg caaggtcgtc gacggtgtcg tgaccgacca gatcgactac 1560 ctgacggctt ccgaagaggt cgacttcaac atcgcgcagg cccc gctcgatgcc 1620 aagggtcgct tccgcgagag ccacgtcctg ccca agggtggcag cggcgaggtc 1680 gacctgttcg tccccgagga gatcggctac atcgacgtct ccccgcgcca gatggtgtcg 1740 gtcgcgacct cgctcgtgcc cttcctcgag cacgacgacg cacagcgcgc cctcatgggt 1800 gccaacatgc agcgtcaggc tgtgccgctg ctgcgcagcg actcgccgct cgtcggaacc 1860 ggtatggagg gctacacggc catcgacgcc ggtgacgtgc tcaccgccga gaaggccggt 1920 gtcgtctccg aggtctccgc agaccgcgtc gtcgtcatgc tcgacgaggg gcag 1980 gagtaccacc tgcgcaagtt cgaccgctcc aaccagggca cgtcgtacaa ccagaaggtc 2040 gtcgtcaccg ccggtgagcg cgtcgaggtc ggagaggtca tcgccgatgg ccccgccacc 2100 gagaacggcg agctggccct cggaaagaac gtcg cgttcatgac gtgggagggc 2160 tacaacttcg aggacgcgat catcctgagc caggacctgg tgaaggacga ctcc 2220 tcgatccaca agta cgaggtcgat gctcgcgaca ccaagctcgg caaggaggag 2280 atcacgcgtg acctccccaa cgtcagcccg gagctgctga aggacctcga cgagcgcggc 2340 atcatccgca ccga ggtccgccct ggcgacatcc tcgtcggcaa ggtcacgccg 2400 aagggtgaga ccgagctgtc ggccgaggag cgcctgctcc gcgcgatctt caacgagaag 2460 agccgcgaag tccgtgacac ctcgctgaag gtgccccacg gtgagcaggg catc 2520 gccgtcaagg agttcaacgc tgaggacggc gacgacgagc tcggctccgg cgtcaaccgc 2580 cgcgtcgtgg tctacatcgc ccagaagcgc aagatcaccg agggtgacaa gctcgccggc 2640 cgtcacggca acaagggtgt catcgcgaag atcctcccga tcgaggacat gccgttcctt 2700 tcggacggta ccccggtcga catcgtgctg aacccgctcg gtatccccgg tcgaatgaac 2760 ttcggtcagg tcctggagac cggg tggatcgcga agcagggctg gaaggtcgag 2820 ggcaacccgg agtgggctgt cccg aaggacgcat tcgaggccgc ccccggcacg 2880 gcca ccccggtgtt cgacggtgcg agcgaggagg agatcgctgg tctcctcgac 2940 gcgaccaccc cgacccgtga cggcgtccgc ctgatcgact cgagcggcaa gacgcagctg 3000 ttcgacggtc gttcgggtga gccgttcccg gcgccgatct ccgtgggcta catgtacatc 3060 ctgc accacctggt cgacgacaag atccacgcac gttccacggg tccgtactcg 3120 atgatcaccc agcagccgct cggtggtaag gcgcagttcg gtggacagcg cttcggtgag 3180 atggaggtgt gggccctcga ggcctacggc gccgcatacg cgctccagga gctcctcacg 3240 atcaagtccg acgacatcct cggccgcgtc aaggtgtacg tcgt caagggcgag 3300 aacatccagg agcccggcat gtcg ttcaaggtgc tcatgaagga gatgcagtcg 3360 ctctgcctga acgtcgaggt cctctcggcc gacggcacgc tggtcaacct ccgcgacacc 3420 gagg cgttccgcgc cgcggaagag atca ccag ccgcttcgag 3480 gccgcctcga agat ctaa 3504 <210> 5 <211> 1167 <212> PRT <213> Microbacterium sp. <220> <221> misc_feature <223> SGI bacterial isolate SGIC06 <220> <221> misc_feature <223> SGI peptide ID T850290 <220> <221> misc_feature <223> RNA-polymerase subunit B <400> 5 Met Ala Ala Ala Arg Asn Ala Ser Thr Ser Thr Thr Thr Lys Asn Gly 1 5 10 15 Arg Gly Ala Ser Arg Leu Ser Phe Ala Lys Ile Ser Asp Thr Leu Thr 25 30 Val Pro Asp Leu Leu Ala Leu Gln Thr Glu Ser Phe Gly Trp Leu Val 40 45 Gly Asn Asp Ala Trp Lys Ala Arg Val Ala Glu Ala Lys Lys Gln Gly 50 55 60 Arg Thr Asp Val Asn Glu Asn Ser Gly Leu Gly Glu Ile Phe Glu Glu 65 70 75 80 Ile Ser Pro Ile Glu Asp Leu Gly Glu Thr Met Gln Leu Ser Phe Thr 85 90 95 Asn Pro Tyr Leu Glu Pro Glu Lys Tyr Ser Ile Glu Glu Cys Lys Glu 100 105 110 Arg Gly Lys Thr Tyr Ala Ala Pro Leu Tyr Val Glu Ala Glu Phe Met 115 120 125 Asn His Leu Thr Gly Glu Ile Lys Thr Gln Thr Val Phe Met Gly Asp 130 135 140 Phe Pro Leu Gln Thr Asp Lys Gly Thr Phe Ile Ile Asn Gly Ser Glu 145 150 155 160 Arg Val Val Val Ser Gln Leu Val Arg Ser Pro Gly Val Tyr Phe Asp 165 170 175 Lys Thr Pro Asp Lys Thr Ser Asp Lys Asp Ile Val Ser Ala Arg Val 180 185 190 Ile Pro Ser Arg Gly Ala Trp Leu Glu Phe Glu Ile Asp Lys Arg Asp 195 200 205 Gln Val Gly Val Arg Val Asp Arg Lys Arg Lys Gln Ser Val Thr Val 210 215 220 Phe Leu Lys Ala Leu Gly Met Thr Ser Glu Glu Ile Leu Ala Glu Phe 225 230 235 240 Ala Gly Tyr Thr Ser Ile Glu Glu Thr Leu Ala Lys Asp Thr Ile Val 245 250 255 Thr Lys Glu Asp Ala Leu Arg Asp Ile Tyr Arg Lys Leu Arg Pro Gly 260 265 270 Glu Gln Val Ala Ala Glu Ala Ala Arg Ala Leu Leu Asp Asn Phe Tyr 275 280 285 Phe Asn Pro Lys Arg Tyr Asp Leu Ala Lys Val Gly Arg Tyr Lys Ile 290 295 300 Asn His Lys Leu Gly Leu Asp Gln Pro Leu Asn Ser Ser Val Leu Thr 305 310 315 320 Val Glu Asp Ile Val Ala Thr Ile Lys Tyr Leu Val Arg Leu His Ala 325 330 335 Gly Thr Glu Glu Thr Phe Thr Gly Ile Arg Gly Gly Lys Lys Ala Glu 340 345 350 Ile Arg Leu Ala Thr Asp Asp Ile Asp Asn Phe Gly Asn Arg Arg Ile 355 360 365 Arg Ala Val Gly Glu Leu Ile Gln Asn Gln Val Arg Thr Gly Leu Ser 370 375 380 Arg Met Glu Arg Val Val Arg Glu Arg Met Thr Thr Gln Asp Ile Glu 385 390 395 400 Ala Ile Thr Pro Gln Thr Leu Ile Asn Val Arg Pro Val Val Ala Ala 405 410 415 Ile Lys Glu Phe Phe Gly Thr Ser Gln Leu Ser Gln Phe Met Asp Gln 420 425 430 Asn Asn Pro Leu Ala Gly Leu Thr Asn Lys Arg Arg Leu Ser Ala Leu 435 440 445 Gly Pro Gly Gly Leu Ser Arg Asp Arg Ala Gly Val Glu Val Arg Asp 450 455 460 Val His Pro Ser His Tyr Gly Arg Met Cys Pro Ile Glu Thr Pro Glu 465 470 475 480 Gly Pro Asn Ile Gly Leu Ile Gly Ala Leu Ala Thr Phe Ala Arg Ile 485 490 495 Asn Ser Phe Gly Phe Ile Glu Thr Pro Tyr Arg Lys Val Val Asp Gly 500 505 510 Val Val Thr Asp Gln Ile Asp Tyr Leu Thr Ala Ser Glu Glu Val Asp 515 520 525 Phe Asn Ile Ala Gln Ala Asn Ala Pro Leu Asp Ala Lys Gly Arg Phe 530 535 540 Arg Glu Ser His Val Leu Ala Arg Pro Lys Gly Gly Ser Gly Glu Val 545 550 555 560 Asp Leu Phe Val Pro Glu Glu Ile Gly Tyr Ile Asp Val Ser Pro Arg 565 570 575 Gln Met Val Ser Val Ala Thr Ser Leu Val Pro Phe Leu Glu His Asp 580 585 590 Asp Ala Gln Arg Ala Leu Met Gly Ala Asn Met Gln Arg Gln Ala Val 595 600 605 Pro Leu Leu Arg Ser Asp Ser Pro Leu Val Gly Thr Gly Met Glu Gly 610 615 620 Tyr Thr Ala Ile Asp Ala Gly Asp Val Leu Thr Ala Glu Lys Ala Gly 625 630 635 640 Val Val Ser Glu Val Ser Ala Asp Arg Val Val Val Met Leu Asp Glu 645 650 655 Gly Gly Thr Gln Glu Tyr His Leu Arg Lys Phe Asp Arg Ser Asn Gln 660 665 670 Gly Thr Ser Tyr Asn Gln Lys Val Val Val Thr Ala Gly Glu Arg Val 675 680 685 Glu Val Gly Glu Val Ile Ala Asp Gly Pro Ala Thr Glu Asn Gly Glu 690 695 700 Leu Ala Leu Gly Lys Asn Leu Leu Val Ala Phe Met Thr Trp Glu Gly 705 710 715 720 Tyr Asn Phe Glu Asp Ala Ile Ile Leu Ser Gln Asp Leu Val Lys Asp 725 730 735 Asp Thr Leu Ser Ser Ile His Ile Glu Glu Tyr Glu Val Asp Ala Arg 740 745 750 Asp Thr Lys Leu Gly Lys Glu Glu Ile Thr Arg Asp Leu Pro Asn Val 755 760 765 Ser Pro Glu Leu Leu Lys Asp Leu Asp Glu Arg Gly Ile Ile Arg Ile 770 775 780 Gly Ala Glu Val Arg Pro Gly Asp Ile Leu Val Gly Lys Val Thr Pro 785 790 795 800 Lys Gly Glu Thr Glu Leu Ser Ala Glu Glu Arg Leu Leu Arg Ala Ile 805 810 815 Phe Asn Glu Lys Ser Arg Glu Val Arg Asp Thr Ser Leu Lys Val Pro 820 825 830 His Gly Glu Gln Gly Thr Ile Ile Ala Val Lys Glu Phe Asn Ala Glu 835 840 845 Asp Gly Asp Asp Glu Leu Gly Ser Gly Val Asn Arg Arg Val Val Val 850 855 860 Tyr Ile Ala Gln Lys Arg Lys Ile Thr Glu Gly Asp Lys Leu Ala Gly 865 870 875 880 Arg His Gly Asn Lys Gly Val Ile Ala Lys Ile Leu Pro Ile Glu Asp 885 890 895 Met Pro Phe Leu Ser Asp Gly Thr Pro Val Asp Ile Val Leu Asn Pro 900 905 910 Leu Gly Ile Pro Gly Arg Met Asn Phe Gly Gln Val Leu Glu Thr His 915 920 925 Leu Gly Trp Ile Ala Lys Gln Gly Trp Lys Val Glu Gly Asn Pro Glu 930 935 940 Trp Ala Val Lys Leu Pro Lys Asp Ala Phe Glu Ala Ala Pro Gly Thr 945 950 955 960 Lys Val Ala Thr Pro Val Phe Asp Gly Ala Ser Glu Glu Glu Ile Ala 965 970 975 Gly Leu Leu Asp Ala Thr Thr Pro Thr Arg Asp Gly Val Arg Leu Ile 980 985 990 Asp Ser Ser Gly Lys Thr Gln Leu Phe Asp Gly Arg Ser Gly Glu Pro 995 1000 1005 Phe Pro Ala Pro Ile Ser Val Gly Tyr Met Tyr Ile Leu Lys Leu 1010 1015 1020 His His Leu Val Asp Asp Lys Ile His Ala Arg Ser Thr Gly Pro 1025 1030 1035 Tyr Ser Met Ile Thr Gln Gln Pro Leu Gly Gly Lys Ala Gln Phe 1040 1045 1050 Gly Gly Gln Arg Phe Gly Glu Met Glu Val Trp Ala Leu Glu Ala 1055 1060 1065 Tyr Gly Ala Ala Tyr Ala Leu Gln Glu Leu Leu Thr Ile Lys Ser 1070 1075 1080 Asp Asp Ile Leu Gly Arg Val Lys Val Tyr Glu Ala Ile Val Lys 1085 1090 1095 Gly Glu Asn Ile Gln Glu Pro Gly Ile Pro Glu Ser Phe Lys Val 1100 1105 1110 Leu Met Lys Glu Met Gln Ser Leu Cys Leu Asn Val Glu Val Leu 1115 1120 1125 Ser Ala Asp Gly Thr Leu Val Asn Leu Arg Asp Thr Asp Asp Glu 1130 1135 1140 Ala Phe Arg Ala Ala Glu Glu Leu Gly Ile Asn Ile Ser Ser Arg 1145 1150 1155 Phe Glu Ala Ala Ser Ile Asp Glu Ile 1160 1165 <210> 6 <211> 1188 <212> DNA <213> Microbacterium sp. <220> <221> misc_feature <223> SGI ial isolate SGIC06 <220> <221> misc_feature <223> SGI Gene ID SG1MICG851004 <220> <221> misc_feature <223> encodes the e ce at SEQ ID NO 7 <400> 6 ttgccgactc gatgtcggtg gcctctccta cggtggaaaa caagccgaag agccattacg ccttgtcgga gagttgctcc caaccgggag cgccgcgaca gcctacaggc ggcaccacgc gcgcagaagg agcacatcat gccatcaccc cgcg agaagtccct cgagaccgcc ctcgcccaga tcgaccgcca gttcggaaag ggctcggtca tgcggctggg cagcgatgag cgcgcccccg tcat cggc tccatcgccc tcgc cctcggcgtc ggaggactcc cgcgtggtcg catcgtcgag atctacggac cggagtcctc cggtaagacg acgctcaccc tgcacgcgat cgcgaacgca cagcgtgccg gtggcatcgc ggcgttcatc gacgccgagc acgcgctcga ccccgactac gccgccaagc tcggcgtcga catcgatgcg ctcctggtct cgcagcccga cacgggtgag caggcgctcg agatcgccga catgctcgtg cgctccggtg cgatcgacct catc gactccgtcg cggccctcgt gccgcgcgcc gagatcgagg gcgagatggg tgactcgcac gtcggtctgc aggctcgcct catgtcgcag gcgctgcgaa agctcaccgg tggtctgaac cagacgaaca ccacgatgat cttcatcaac cagctccgcg agaagatcgg tgtcttcttc ggttcgccgg agaccactgc cggcggtaag gcgctcaagt tctacgcctc ggtccgcatg gacatccgtc gtatcgagac gctcaaggac ggtactgacg ctgtcggtaa ccgcaccagg gtcaaggtcg tcaagaacaa gatggctccg cctttcaagc aggccgagtt cgacatcctc tacggcgtcg gcatctcgcg cgagggaagc ctgatcgact tcggtgtcga gcatgcgatc gtcaagaagt cctg gtac 1020 gacggtgacc agctgggtca gggcaaggag aacgcgcgga cgttcctgct caacaacccc 1080 gacatcgcgc tggcgatcga gacgcagatc aagcagaagc tcggcatcgg cggtcccgcc 1140 gcggcgcctg ctgcggcaga cgct gagcgtcgtc cggcctga 1188 <210> 7 <211> 395 <212> PRT <213> Microbacterium sp. <220> <221> misc_feature <223> SGI bacterial isolate SGIC06 <220> <221> misc_feature <223> SGI peptide ID SG1MICT851004 <220> <221> misc_feature <223> recombinase A <400> 7 Met Pro Thr Arg Cys Arg Trp Pro Leu Leu Arg Trp Lys Thr Ser Arg 1 5 10 15 Arg Ala Ile Thr Pro Cys Arg Arg Val Ala Pro Asn Arg Glu Arg Arg 25 30 Asp Ser Leu Gln Ala Ala Pro Arg Ala Gln Lys Glu His Ile Met Pro 40 45 Ser Pro Ala Asp Arg Glu Lys Ser Leu Glu Thr Ala Leu Ala Gln Ile 50 55 60 Asp Arg Gln Phe Gly Lys Gly Ser Val Met Arg Leu Gly Ser Asp Glu 65 70 75 80 Arg Ala Pro Val Ala Val Ile Pro Thr Gly Ser Ile Ala Leu Asp Val 85 90 95 Ala Leu Gly Val Gly Gly Leu Pro Arg Gly Arg Ile Val Glu Ile Tyr 100 105 110 Gly Pro Glu Ser Ser Gly Lys Thr Thr Leu Thr Leu His Ala Ile Ala 115 120 125 Asn Ala Gln Arg Ala Gly Gly Ile Ala Ala Phe Ile Asp Ala Glu His 130 135 140 Ala Leu Asp Pro Asp Tyr Ala Ala Lys Leu Gly Val Asp Ile Asp Ala 145 150 155 160 Leu Leu Val Ser Gln Pro Asp Thr Gly Glu Gln Ala Leu Glu Ile Ala 165 170 175 Asp Met Leu Val Arg Ser Gly Ala Ile Asp Leu Ile Val Ile Asp Ser 180 185 190 Val Ala Ala Leu Val Pro Arg Ala Glu Ile Glu Gly Glu Met Gly Asp 195 200 205 Ser His Val Gly Leu Gln Ala Arg Leu Met Ser Gln Ala Leu Arg Lys 210 215 220 Leu Thr Gly Gly Leu Asn Gln Thr Asn Thr Thr Met Ile Phe Ile Asn 225 230 235 240 Gln Leu Arg Glu Lys Ile Gly Val Phe Phe Gly Ser Pro Glu Thr Thr 245 250 255 Ala Gly Gly Lys Ala Leu Lys Phe Tyr Ala Ser Val Arg Met Asp Ile 260 265 270 Arg Arg Ile Glu Thr Leu Lys Asp Gly Thr Asp Ala Val Gly Asn Arg 275 280 285 Thr Arg Val Lys Val Val Lys Asn Lys Met Ala Pro Pro Phe Lys Gln 290 295 300 Ala Glu Phe Asp Ile Leu Tyr Gly Val Gly Ile Ser Arg Glu Gly Ser 305 310 315 320 Leu Ile Asp Phe Gly Val Glu His Ala Ile Val Lys Lys Ser Gly Ser 325 330 335 Trp Tyr Thr Tyr Asp Gly Asp Gln Leu Gly Gln Gly Lys Glu Asn Ala 340 345 350 Arg Thr Phe Leu Leu Asn Asn Pro Asp Ile Ala Leu Ala Ile Glu Thr 355 360 365 Gln Ile Lys Gln Lys Leu Gly Ile Gly Gly Pro Ala Ala Ala Pro Ala 370 375 380 Ala Ala Asp Glu Leu Ala Glu Arg Arg Pro Ala 385 390 395 <210> 8 <211> 2175 <212> DNA <213> acterium sp. <220> <221> misc_feature <223> SGI bacterial isolate SGIC06 <220> <221> misc_feature <223> SGI Gene ID SG1MICG849768 <220> <221> misc_feature <223> encodes the peptide sequence at SEQ ID NO 9 <400> 8 atgtacccca tgatcgatcc cgcactcgcc ggcc acgc cgaagacgac gacttcgacg agtc tcccgactcc cagctcccgg accaccgcta cctggatcgc gagctgagct ggctcgcctt caaccagcgc gagc tcgccgagga tccgtcactg ctcg cgaa cttcctggcg atcttcgcca gcaacctcga cgagttcttc atggtgcgcg tcgccggcct caagcgccgc atcatgaccg gcctggccgt gccgacgaac atcggccgct cccccgtcga cgcgctcgcc gacatctccc gcgaagcgca cgccctgcag ctgcgtcacg ccgaggcctg gacctcgctc cccg ccctggccga ctccggcatc gagatcacgg actggtccga gctgaccgac gacgagcgct ccggattgtc cgagtacttc cagctccagg cggt gccg ctcgcggtcg acccggcgca tccgttcccc tacatctccg gcctgtcgct gaacctcgcg atccgcatcc gcaatgcccg caccgggcgc caggagttcg cgcgcctcaa ggtgccgccc atgctccccc gcttcgtgga ggtgccgggc ggcggcgaga tcaagcgctt cctgcgcctg gaggaactga tcgcgaacca cctcggcgac ctgttccccg gcatggaggt gctcgaccac cacgcgttcc gcctcacccg caacgaagac gtggagatcg aggaagacga gagcgagaac ctcatccagg aggc cgagctgctg cgccgtcgat tcggcccgcc gatccgcctc gagatcacgg acgacatgga cgaggtcacg ctgc tcgtccgcga gctcgacatc accgacctgg aggtctaccg cctccccggt ccgctcgacc tgcgcggact gttcgatctg tcccgcatcg accgtcccga cctgcgctac 1020 ccgccgcacc tgcccaccac ggccgtggcc ttccagcccg caggatcgag caaccgcgcc 1080 ttca aagcgatccg caagtcggat gtgctcgtgc accacccgta gttc 1140 agcg tgcaggcgtt cctcgaacag gccgcccgcg acccgcacgt gctcgccatc 1200 aagcagaccc tgtaccgcac ctcgggcgac agcccggtcg tgcaggcgct gatcgacgcg 1260 gccgaagccg gcaagcaggt gctggccctc gtcgaggtga aggcccgttt cgacgaggcc 1320 aacaacatcg tctgggcacg caagctcgag aaggccggcg tgcacgtggt ctacggtctc 1380 gtcggactca agacccactg cgcc ctcgtcatcc gcgaggaaga ggggatgctg 1440 cgccactact cgcacgtcgg caccggcaac tacaacccca agaccagccg catctacgag 1500 gacttcggtc ccgc agacgcgcag gtcggcaaag acctgacacg cctgttcaac 1560 gagctcagcg gctacgcgat cgagaagaag ttcaagcgcc tgctggtcgc cccgctgcac 1620 aagg tccg ccagatcgac cgca ggaacgccga ggcggggatc 1680 cccgcgcaca tccgcatcaa ggtgaactcg atggtcgatg aggagatcat cgacgcgctc 1740 taccgcgcga gcgcggccgg ggtgaaggtc gacgtgtggg tgcgcggcat ctgcagcctg 1800 cgcaccgacc tcgacggcat cagtgacaac atcacggtgc gcagcatcct cggccgctac 1860 ctcgagcact cccgcatctt cgag aacgccggcg acccgcaggt gtacatcggc 1920 agcgccgaca accg caacctcgac cgtcgtgtgg aggcgctggt cacc 1980 gacgccgacc acctcaagga actgcaggcg ttcttcgacc tcgcgatgga cgacggaacc 2040 tcgtcgtggc atctcggcgc cggcggcgtc tgggagcgcc acgccgtgaa cgccgacggc 2100 aagccgctga tcgacctgca ggataagacc atggggttga tccagcggcg ccgccgcgcg 2160 cgggcggttc gatga 2175 <210> 9 <211> 724 <212> PRT <213> Microbacterium sp. <220> <221> misc_feature <223> SGI bacterial isolate SGIC06 <220> <221> misc_feature <223> SGI e ID SG1MICT849768 <220> <221> misc_feature <223> polyphosphate kinase <400> 9 Met Tyr Pro Met Ile Asp Pro Ala Leu Ala Asp Ala Gly Leu Gly Asp 1 5 10 15 Ala Glu Asp Asp Asp Phe Asp Ala Ile Glu Ser Pro Asp Ser Gln Leu 25 30 Pro Asp His Arg Tyr Leu Asp Arg Glu Leu Ser Trp Leu Ala Phe Asn 40 45 Gln Arg Val Met Glu Leu Ala Glu Asp Pro Ser Leu Pro Glu Leu Glu 50 55 60 Arg Ala Asn Phe Leu Ala Ile Phe Ala Ser Asn Leu Asp Glu Phe Phe 65 70 75 80 Met Val Arg Val Ala Gly Leu Lys Arg Arg Ile Met Thr Gly Leu Ala 85 90 95 Val Pro Thr Asn Ile Gly Arg Ser Pro Val Asp Ala Leu Ala Asp Ile 100 105 110 Ser Arg Glu Ala His Ala Leu Gln Leu Arg His Ala Glu Ala Trp Thr 115 120 125 Ser Leu Val Arg Pro Ala Leu Ala Asp Ser Gly Ile Glu Ile Thr Asp 130 135 140 Trp Ser Glu Leu Thr Asp Asp Glu Arg Ser Gly Leu Ser Glu Tyr Phe 145 150 155 160 Gln Leu Gln Val Phe Pro Val Leu Met Pro Leu Ala Val Asp Pro Ala 165 170 175 His Pro Phe Pro Tyr Ile Ser Gly Leu Ser Leu Asn Leu Ala Ile Arg 180 185 190 Ile Arg Asn Ala Arg Thr Gly Arg Gln Glu Phe Ala Arg Leu Lys Val 195 200 205 Pro Pro Met Leu Pro Arg Phe Val Glu Val Pro Gly Gly Gly Glu Ile 210 215 220 Lys Arg Phe Leu Arg Leu Glu Glu Leu Ile Ala Asn His Leu Gly Asp 225 230 235 240 Leu Phe Pro Gly Met Glu Val Leu Asp His His Ala Phe Arg Leu Thr 245 250 255 Arg Asn Glu Asp Val Glu Ile Glu Glu Asp Glu Ser Glu Asn Leu Ile 260 265 270 Gln Ala Leu Glu Ala Glu Leu Leu Arg Arg Arg Phe Gly Pro Pro Ile 275 280 285 Arg Leu Glu Ile Thr Asp Asp Met Asp Glu Val Thr Met Asp Leu Leu 290 295 300 Val Arg Glu Leu Asp Ile Thr Asp Leu Glu Val Tyr Arg Leu Pro Gly 305 310 315 320 Pro Leu Asp Leu Arg Gly Leu Phe Asp Leu Ser Arg Ile Asp Arg Pro 325 330 335 Asp Leu Arg Tyr Pro Pro His Leu Pro Thr Thr Ala Val Ala Phe Gln 340 345 350 Pro Ala Gly Ser Ser Asn Arg Ala Asp Ile Phe Lys Ala Ile Arg Lys 355 360 365 Ser Asp Val Leu Val His His Pro Tyr Glu Ser Phe Thr Thr Ser Val 370 375 380 Gln Ala Phe Leu Glu Gln Ala Ala Arg Asp Pro His Val Leu Ala Ile 385 390 395 400 Lys Gln Thr Leu Tyr Arg Thr Ser Gly Asp Ser Pro Val Val Gln Ala 405 410 415 Leu Ile Asp Ala Ala Glu Ala Gly Lys Gln Val Leu Ala Leu Val Glu 420 425 430 Val Lys Ala Arg Phe Asp Glu Ala Asn Asn Ile Val Trp Ala Arg Lys 435 440 445 Leu Glu Lys Ala Gly Val His Val Val Tyr Gly Leu Val Gly Leu Lys 450 455 460 Thr His Cys Lys Leu Ala Leu Val Ile Arg Glu Glu Glu Gly Met Leu 465 470 475 480 Arg His Tyr Ser His Val Gly Thr Gly Asn Tyr Asn Pro Lys Thr Ser 485 490 495 Arg Ile Tyr Glu Asp Phe Gly Leu Phe Thr Ala Asp Ala Gln Val Gly 500 505 510 Lys Asp Leu Thr Arg Leu Phe Asn Glu Leu Ser Gly Tyr Ala Ile Glu 515 520 525 Lys Lys Phe Lys Arg Leu Leu Val Ala Pro Leu His Leu Arg Lys Gly 530 535 540 Leu Ile Arg Gln Ile Asp Ala Glu Arg Arg Asn Ala Glu Ala Gly Ile 545 550 555 560 Pro Ala His Ile Arg Ile Lys Val Asn Ser Met Val Asp Glu Glu Ile 565 570 575 Ile Asp Ala Leu Tyr Arg Ala Ser Ala Ala Gly Val Lys Val Asp Val 580 585 590 Trp Val Arg Gly Ile Cys Ser Leu Arg Thr Asp Leu Asp Gly Ile Ser 595 600 605 Asp Asn Ile Thr Val Arg Ser Ile Leu Gly Arg Tyr Leu Glu His Ser 610 615 620 Arg Ile Phe Ala Phe Glu Asn Ala Gly Asp Pro Gln Val Tyr Ile Gly 625 630 635 640 Ser Ala Asp Met Met His Arg Asn Leu Asp Arg Arg Val Glu Ala Leu 645 650 655 Val Arg Val Thr Asp Ala Asp His Leu Lys Glu Leu Gln Ala Phe Phe 660 665 670 Asp Leu Ala Met Asp Asp Gly Thr Ser Ser Trp His Leu Gly Ala Gly 675 680 685 Gly Val Trp Glu Arg His Ala Val Asn Ala Asp Gly Lys Pro Leu Ile 690 695 700 Asp Leu Gln Asp Lys Thr Met Gly Leu Ile Gln Arg Arg Arg Arg Ala 705 710 715 720 Arg Ala Val Arg <210> 10 <211> 1390 <212> DNA <213> Microbacterium sp. <220> <221> misc_feature <223> SGI bacterial isolate SGIG08 <220> <221> misc_feature <223> encodes a 16S mal RNA <220> <221> misc_feature <222> (29)..(36) <223> n is a, c, g, t, or other <220> <221> misc_feature <222> (38)..(41) <223> n is a, c, g, t, or other <400> 10 aacggtgaac acggagcttg ctctgtggnn nnnnnngnnn gagt aacacgtgag caacctgccc ctgactctgg gataagcgct ggaaacggcg tctaatactg gatacgagta gcgatcgcat ggtcagctac tggaaagatt ttttggttgg ggatgggctc gcggcctatc agcttgttgg tgaggtaatg gctcaccaag gcgtcgacgg gtagccggcc tgagagggtg accggccaca ctgggactga gacacggccc ctac gggaggcagc agtggggaat attgcacaat gggcggaagc ctgatgcagc aacgccgcgt gagggatgac ggccttcggg ttgtaaacct gcag ggaagaagcg aaagtgacgg tacctgcaga aaaagcgccg gctaactacg cagc cgcggtaata cgtagggcgc aagcgttatc cggaattatt gggcgtaaag agctcgtagg cggtttgtcg cgtctgctgt gaaatcccga ggctcaacct cgggcctgca gtgggtacgg gcagactaga gtgcggtagg ggagattgga attcctggtg tgga atgcgcagat atcaggagga atgg cgaaggcaga tctctgggcc gtaactgacg ctgaggagcg aaagggtggg gagcaaacag gcttagatac cctggtagtc caccccgtaa acgttgggaa ctagttgtgg ggtccattcc acggattccg tgacgcagct aacgcattaa gttccccgcc tggggagtac ggccgcaagg ctaaaactca aaggaattga cccg cacaagcggc ggagcatgcg gattaattcg atgcaacgcg aagaacctta ccaaggcttg acga gaacgggcca gaaatggtca actctttgga cactcgtaaa caggtggtgc atggttgtcg cgtg tcgtgagatg ttgggttaag tcccgcaacg 1020 agcgcaaccc tcgttctatg ttgccagcac gtaatggtgg gaactcatgg gatactgccg 1080 gggtcaactc aggt ggggatgacg tcaaatcatc atgcccctta tgtcttgggc 1140 ttcacgcatg ctacaatggc cggtacaaag ggctgcaata ccgtgaggtg gagcgaatcc 1200 gccg gtcccagttc ggattgaggt ctgcaactcg acctcatgaa gtcggagtcg 1260 ctagtaatcg cagatcagca acgctgcggt gaatacgttc ccgggtcttg tacacaccgc 1320 ccgtcaagtc atgaaagtcg cctg aagccggtgg cctt gtggagggag 1380 ccgtcgaagg 1390 <210> 11 <211> 532 <212> DNA <213> Mycosphaerella sp. <220> <221> eature <223> SGI bacterial isolate SGIH11 <220> <221> misc_feature <223> Internal transcribed spacer 1_5.8S ribosomal DNA <220> <221> misc_feature <222> (43)..(43) <223> n is a, c, g, or t <220> <221> misc_feature <222> (526)..(526) <223> n is a, c, g, or t <400> 11 cggagggatc attaatgagt gagggggcca cccccaacct ccnacccttt gtgaacgcat catgttgctt cgggggcgac gttc gcggcattcc ccccggaggt catcaaaaca ctgcattctt acgtcggagt aaaaagttaa tttaataaaa ctttcaacaa cggatctctt ggttctggca aaga acgcagcgaa atgcgataag taatgtgaat attc agtgaatcat cgaatctttg aacgcacatt gcgccccctg gtattccggg gggcatgcct gttcgagcgt catttcacca ctcaagcctc gcttggtatt gggcgtcgcg agtctctcgc gcgcctcaaa gtctccggct gttcggttcg agcg ttgtggcaac tatttcgcag tggagtacga gtcgtggcgg aatc tttcaaaggt tgacctcgga tcaggtaggg atacccgctg aacttaagca tatcaataag cggaggaggt catagntgtt tc <210> 12 <211> 1431 <212> DNA <213> Variovorax sp. <220> <221> misc_feature <223> SGI bacterial isolate SGIG01 <220> <221> misc_feature <223> encodes a 16S ribosomal RNA <400> 12 tgccttacac atgcaagtcg aacggcagcg cgggagcaat cctggcggcg agtggcgaac gggtgagtaa tacatcggaa cgtgcccaat cgtgggggat aacgcagcga aagctgtgct aataccgcat acgatctacg gatgaaagca ggggatcgca agaccttgcg cgaatggagc ggccgatggc agattaggta gttggtgagg taaaggctca ccaagccttc gatctgtagc tggtctgaga ccag ccacactggg actgagacac ggcccagact cctacgggag gcagcagtgg ggaattttgg acaatgggcg aaagcctgat ccagccatgc cagg atgaaggcct tcgggttgta aactgctttt gtacggaacg aaacggcctt ttctaataaa taat accg taagaataag caccggctaa gcca gcagccgcgg taatacgtag ggtgcaagcg ttaatcggaa ttactgggcg taaagcgtgc gcaggcggtt atgtaagaca gttgtgaaat ccccgggctc aacctgggaa ctgt gactgcatag ctagagtacg gtagaggggg atggaattcc gcgtgtagca tgcg tagatatgcg gaggaacacc gatggcgaag gcaatcccct ggacctgtac tgacgctcat gcacgaaagc gtggggagca aacaggatta gataccctgg tagtccacgc cctaaacgat gtcaactggt gtct tcactgactc agtaacgaag ctaacgcgtg aagttgaccg cctggggagt acggccgcaa ggttgaaact caaaggaatt gacggggacc cgcacaagcg gtggatgatg tggtttaatt cgatgcaacg cgaaaaacct tacccacctt tgacatgtac ggaattcgcc agagatggct tagtgctcga aagagaaccg taacacaggt gctgcatggc tgtcgtcagc 1020 cgtg agatgttggg cccg caacgagcgc tgtc attagttgct 1080 acattcagtt gggcactcta atgagactgc cggtgacaaa ccggaggaag gtggggatga 1140 cgtcaagtcc tcatggccct tataggtggg gctacacacg tcatacaatg gctggtacaa 1200 agggttgcca acccgcgagg gggagctaat cccataaaac tagt ccggatcgca 1260 gtctgcaact cgactgcgtg aagtcggaat cgctagtaat cgtggatcag aatgtcacgg 1320 tgaatacgtt tctt gtacacaccg acac catgggagcg ggttctgcca 1380 gaagtagtta gcttaaccgc aaggagggcg attaccacgg cagggttcgt g 1431 <210> 13 <211> 1424 <212> DNA <213> Bacillus amyloliquefaciens <220> <221> eature <223> SGI bacterial isolate SGIF03 <220> <221> misc_feature <223> encodes a 16S ribosomal RNA <400> 13 ctggcggcgt gcctaataca tgcaagtcga gcggacagat gggagcttgc tccctgatgt tagcggcgga cgggtgagta acacgtgggt aacctgcctg taagactggg ataactccgg gaaaccgggg ctaataccgg atggttgtct catg gttcagacat tggc ttcggctacc acttacagat ggacccgcgg cgcattagct agttggtgag gtaacggctc gcga cgatgcgtag ccgacctgag agggtgatcg ctgg gactgagaca cggcccagac tcctacggga agta gggaatcttc cgcaatggac gaaagtctga cggagcaacg ccgcgtgagt gatgaaggtt ttcggatcgt ctgt tgttagggaa gaacaagtgc cgttcaaata gggcggcacc ttgacggtac ctaaccagaa agccacggct aactacgtgc cagcagccgc ggtaatacgt aggtggcaag cgttgtccgg aattattggg cgtaaagggc tcgcaggcgg tttcttaagt ctgatgtgaa agcccccggc gggg agggtcattg gaaactgggg aacttgagtg agga gagtggaatt ccacgtgtag cggtgaaatg cgtagagatg tggaggaaca ccagtggcga aggcgactct ctggtctgta actgacgctg aggagcgaaa gcgtggggag cgaacaggat tagataccct ggtagtccac gccgtaaacg atgagtgcta agtgttaggg ggtttccgcc ccttagtgct gcagctaacg cattaagcac tccgcctggg ggtc gcaagactga aactcaaagg aattgacggg ggcccgcaca ggag catgtggttt aattcgaagc aacgcgaaga accttaccag gtcttgacat cctctgacaa tcctagagat aggacgtccc cttcgggggc agagtgacag 1020 gtggtgcatg gttgtcgtca gctcgtgtcg tgagatgttg ggttaagtcc cgcaacgagc 1080 gcaacccttg atcttagttg ccagcattca cact ctaaggtgac tgac 1140 aaaccggagg aaggtgggga tgacgtcaaa tcatcatgcc ccttatgacc tgggctacac 1200 acgtgctaca atggacagaa caaagggcag cgaaaccgcg aggttaagcc aatcccacaa 1260 atctgttctc agttcggatc gcagtctgca actcgactgc gtgaagctgg aatcgctagt 1320 aatcgcggat cagcatgccg cggtgaatac gttcccgggc cttgtacaca ccgcccgtca 1380 caccacgaga aaca cccgaagtcg gtgaggtaac cttt 1424 <210> 14 <211> 1425 <212> DNA <213> Bacillus subtilis <220> <221> misc_feature <223> SGI bacterial isolate SGIH06 <220> <221> misc_feature <223> s a 16S ribosomal RNA <400> 14 gctggcggcg tgcctaatac atgcaagtcg agcggacaga cttg ctccctgatg ttagcggcgg gagt aacacgtggg taacctgcct gtaagactgg gataactccg ggaaaccggg gctaataccg gatggttgtt tgaaccgcat ggttcagaca taaaaggtgg cttcggctac cacttacaga tggacccgcg gcgcattagc tagttggtga ggtaacggct caccaaggca acgatgcgta gccgacctga gagggtgatc ggccacactg ggactgagac acggcccaga ctcctacggg aggcagcagt agggaatctt ccgcaatgga tctg acggagcaac gccgcgtgag tgatgaaggt tttcggatcg taaagctctg ttgttaggga agaacaagtg ccgttcaaat gcac cttgacggta cctaaccaga aagccacggc taactacgtg ccagcagccg cggtaatacg taggtggcaa gcgttgtccg gaattattgg gcgtaaaggg ggcg gtttcttaag tctgatgtga aagcccccgg ctcaaccggg gagggtcatt ggaaactggg gaacttgagt gagg agagtggaat tgta gcggtgaaat gcgtagagat gtggaggaac accagtggcg aaggcgactc tctggtctgt aactgacgct gaggagcgaa agcgtgggga gcgaacagga ttagataccc tcca cgccgtaaac gatgagtgct aagtgttagg gggtttccgc cccttagtgc tgcagctaac gcattaagca ctccgcctgg ggagtacggt cgcaagactg aaactcaaag acgg gggcccgcac tgga gcatgtggtt taattcgaag caacgcgaag aaccttacca gaca tcctctgaca atcctagaga taggacgtcc ccttcggggg cagagtgaca 1020 ggtggtgcat ggttgtcgtc agctcgtgtc tgtt gggttaagtc ccgcaacgag 1080 cgcaaccctt gatcttagtt gccagcattc agttgggcac tctaaggtga ctgccggtga 1140 caaaccggag gaaggtgggg atgacgtcaa atcatcatgc cccttatgac ctgggctaca 1200 cacgtgctac aatggacaga acaaagggca gcgaaaccgc gaggttaagc caca 1260 aatctgttct cagttcggat cgcagtctgc aactcgactg cgtgaagctg gaatcgctag 1320 taatcgcgga tcagcatgcc gcggtgaata cgttcccggg ccttgtacac accgcccgtc 1380 acaccacgag agtttgtaac acccgaagtc ggtgaggtaa ccttt 1425 <210> 15 <211> 37 <212> DNA <213> Artificial ce <220> <223> PCR primer M13-ITS1 <400> 15 tgtaaaacga cggccagttt cgtaggtgaa cctgcgg <210> 16 <211> 38 <212> DNA <213> Artificial ce <220> <223> PCR primer ITS4-M13 <400> 16 caggaaacag ctatgacctc ctccgcttat tgatatgc <210> 17 <211> 39 <212> DNA <213> Artificial sequence <220> <223> PCR primer M13-27F Bac <400> 17 tgtaaaacga cggccagtta gagtttgatc ctggctcag <210> 18 <211> 37 <212> DNA <213> Artificial sequence <220> <223> PCR primer 1492R-M13 Bac <400> 18 caggaaacag ctatgaccgg tgtt acgactt

Claims (22)

1. An isolated microbial strain of Bacillus sp. strain 5-F03 (deposited as NRRL B- 50760) and pesticidally active variants thereof, wherein said ial strain has suppressive activity against head blight disease.

2. An isolated microbial strain ing to claim 1, wherein said microbial strain comprises a DNA sequence exhibiting at least 85% sequence identity to any one of the tide sequences in the Sequence Listing.

3. A biologically pure culture of a microbial strain according to claim 1.

4. An enriched culture of a microbial strain ing to claim 1.

5. A composition comprising a microbial strain or culture thereof of any one of claims 1- 4, and an agriculturally effective amount of a compound or composition selected from the group consisting of an acaricide, a bactericide, a ide, an insecticide, a microbicide, a nematicide, a pesticide, and a fertilizer.

6. A composition comprising a microbial strain or culture thereof of any one of claims 1- 4, and a carrier.

7. A composition according to claim 6, wherein said carrier is an agriculturally acceptable carrier.

8. A composition according to claim 6, n said carrier is a plant seed.

9. A composition according to claim 6, wherein said composition is prepared as a formulation selected from the group consisting of an emulsion, a colloid, a dust, a granule, a pellet, a powder, a spray, an emulsion, and a solution.

10. A composition ing to claim 6, wherein said composition is a seed coating formulation.

11. A seed having a coating comprising a composition according to claim 6.

12. A method for preventing, inhibiting or treating the development of a plant pathogen, wherein said method comprises growing a microbial strain or culture thereof of any one of claims 1-4 in a growth medium or soil of a host plant prior to or rent with host plant growth in said growth medium or soil.

13. A method according to claim 12, wherein said plant pathogen causes head blight disease.

14. A method ing to claim 13, wherein said plant pathogen is Fusarium graminearum.

15. A method for preventing, inhibiting or treating the pment of head blight disease of a plant, wherein said method comprises applying to the plant, or to the plant's surroundings, an effective amount of a microbial strain or culture thereof of any one of claims 1-4.

16. A method according to claim 15, n said ial strain or culture thereof is applied to soil, a seed, a root, a flower, a leaf, a portion of the plant, or the whole plant.

17. A method according to claim 15, wherein said plant is susceptible to um graminearum.

18. A method according to claim 15, wherein said plant is a wheat plant, a corn plant, a barley plant, or an oat plant.

19. A method according to claim 15, wherein said microbial strain or culture thereof is established as an endophyte on said plant.

20. A non-naturally occurring plant that is a plant cially infected with a microbial strain or culture thereof of any one of claims 1-4.

21. Seed, reproductive tissue, vegetative tissue, plant parts, or progeny of a non-naturally occurring plant of claim 20.

22. A method for preparing an agricultural ition, wherein said method comprises inoculating a ial strain or culture thereof according to any one of claims 1-4 into or onto a substratum and allowing said microbial strain or culture thereof to grow at a temperature of 1-37°C until obtaining a number of cells or spores of at least 102-103 per milliliter or per gram.